In vitro diagnostic devices comprising beads and uses thereof

Abstract

The present invention relates to an in vitro diagnostic device for detecting and / or identifying antigens and / or antibodies from a biological fluid sample, in particular from a blood sample or a blood component sample. The present invention also relates to different uses of the device, such as detecting and / or identifying red blood cell antigens, platelet antigens, viral antigens, bacterial antigens, parasite antigens, detecting and / or identifying anti-red blood cell antibodies, anti-platelet antibodies, anti-viral antibodies, anti-bacterial antibodies and anti-parasite antibodies.

Description

Technical Field

[0001] The present invention belongs to the field of immunodiagnosis. In particular, the present invention relates to an in vitro diagnostic device for detecting and / or identifying antigens and / or antibodies from a biological fluid sample, in particular from a blood sample or a blood component sample. The present invention also relates to various uses of the device, such as detecting and / or identifying 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, parasite antigens or anti-parasite antibodies. In vitro methods using the device of the present invention are also part of the present invention. Background Art

[0002] Immunodiagnostic assays are methods that can be used in a variety of test formats and can be used to detect antibodies, antigens, or a combination of both. Typically, these detection tests are based on the use of fixed antigens that capture any specific antibodies present in the sample being tested, while tests for detecting antigens consist on fixed antibodies that allow the capture of specific antigens present in the sample.

[0003] These analyses are widely used, for example, in immunohematology, for the detection of infectious (infectious) pathogens, among other things, and especially to ensure the safety of blood transfusions.

[0004] A blood transfusion consists in the intravenous administration of a concentrated red blood cell preparation (globulin concentrate) obtained from the donor's blood. There are two main risks of a blood transfusion:

[0005] The most important risk is the possible aggregation of antibodies and their erythrocyte antigens in the body of the recipient (the person receiving the transfusion). The consequences of such an immunological reaction can range from ineffective transfusion with no clinical symptoms to mild clinical reactions (anxiety, tremors), severe clinical reactions (shock, hemoglobinuria, renal failure) or violent clinical reactions leading to death (shock, disseminated intravascular hemolysis).

[0006] Blood transfusion is a possible source of disease transmission. Numerous agents can potentially be transmitted through blood transfusion, including bacteria, viruses and parasites. Of these, bacteria are the most commonly transmitted and include species of the following genera: Treponema, Yersinia, Proteus, Pseudomonas, Escherichia, Klebsiella, Acinetobacter and Serratia, while among Gram-positive organisms, Propionibacterium, Staphylococcus, Bacillus, Clostridium and Enterococcus have been isolated. Viral sources that can be transmitted through blood transfusion include human immunodeficiency virus (HIV), hepatitis viruses, West Nile virus (WNV), cytomegalovirus (CMV), human T-cell lymphotropic virus (HTLV), and parvovirus B 19. Another example is protozoan organisms, which include species of the genus Plasmodium, which cause malaria and can be transmitted through blood transfusion.

[0007] To ensure the safety of blood transfusion, on the one hand, the donor's red blood cells are required to be compatible with the recipient's blood if the recipient has no circulating antibodies against the donor's red blood cell antigens, and on the other hand, the donor's red blood cells are required to be free of transfusion-transmissible infectious agents.

[0008] To ensure compatibility between donor red blood cells and recipient, tests for blood typing and the presence of anti-red blood cell antibodies in plasma / serum are currently used.

[0009] The purpose of immunohematological diagnosis is to measure and / or identify the immune reaction between red blood cell antigens and antibodies specific for such antigens. To this end, it is necessary to have tools for determining the antigens present at 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 against known antigens of the donor's red blood cells, the presence of which indicates the possibility of incompatibility.

[0010] To date, among all the antigenic variants of the erythrocyte membrane antigens constituting the blood group, more than thirty erythrocyte antigen systems have been identified in humans: the ABO system with A (ABO1), B (ABO2), AB (AB03) and A1 (ABO4), the Rh system with D (RH1), E (Rh3) or e (RH5) and C (Rh2) or c (RH4) antigens, the Kell system (K or KEL1, k or KEL2...), the Duffy system (Fya or FY1, Fyb or FY2...), the Kidd system (Jka or JK1, Jkb or JK2...), the MNS (MNS1, MNS2, MNS3, MNS4...) system or other systems that are less studied in practice, such as Lutheran, Lewis, etc.... Individuals with the same combination of erythrocyte antigens belong to the same erythrocyte blood group. When several antigen systems are used, blood group becomes more complex and numerous.

[0011] Conventional techniques consist in searching for and identifying the presence or absence of red blood cell antigens at the surface of these cells and / or searching for and identifying the presence or absence of anti-red blood cell antibodies of the blood group in plasma or serum.

[0012] For example, for the ABO system, the Beth-Vincent test measures antigens carried by red blood cells, while the complementary Simonin-Michon test or reverse blood grouping measures antibodies circulating in serum or plasma.

[0013] In the Beth-Vincent test, an individual's red blood cells are combined with an antibody reagent of known specificity. Typically, the test is visualized by observing the agglutination of the red blood cells when the antibody reagent recognizes the corresponding red blood cell antigen.

[0014] In the Simonin-Michon test, the plasma or serum of an individual is combined with a red blood cell test substance, each of which belongs to a precise group of antigens of the ABO system. This is a test of the agglutination of red blood cells from antibodies present in the plasma or serum of an individual. These antibodies of the IgM type immunoglobulin are able to agglutinate red blood cells in vitro. They are called "regular" antibodies because they are systematically present in the serum or plasma of individuals who do not carry the corresponding antigens at the surface of their red blood cells.

[0015] There is also a second class of antibodies known as "irregular" (or "immune") antibodies. Their presence in serum or plasma is optional and they are directed against antigens that are not of the ABO system. This usually involves IgG (sometimes IgM), which appear during antigenic stimulation of foreign red blood cells, for example after a blood transfusion or during pregnancy by a maternal immune response to fetal red blood cell antigens that do not belong to the maternal blood group, or after immunization against one or more antigens after an allogeneic transplant.

[0016] The screening of these irregular antibodies is called the indirect agglutinin test (IAT) or indirect Coombs analysis. This test is used to detect the presence or absence of IgG antibodies against various red blood cell antigens in the blood of an individual by promoting agglutination using human antiglobulins, since IgG itself is not able to spontaneously induce agglutination in vitro. For this reason, the test is intended to demonstrate the binding of these antibodies on test red blood cells of known antigenicity. The method is performed on several red blood cells with different known antigenicities at the same time, and comparison of the results allows identification of the specificity of the IgG present.

[0017] For most immunogenic antigens, such as Rh D (RH1), the risk is greater, but it is also true for other rhesus macaque types (E(RH3)>c(RH4)>e(RH5)>C(RH3)), antigens of the Kell system (KEL1), antigens of the Duffy system (FY1, FY2), antigens of the Kidd system (JK1, JK2), etc...

[0018] In practice, it is not possible to take all these antigens into account when performing a transfusion, since it is impossible to obtain the right blood type at the right moment, especially since some antigen combinations are extremely rare. Standard transfusions take into account only the ABO blood type and the presence or absence of antigen D (RH:1 or RH:-1). In the presence of irregular antibodies, many other systems are taken into account, especially 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 blood type of the donor and the blood type of the recipient by taking into account the presence or risk of occurrence of these irregular anti-red blood cell antibodies.

[0019] Therefore, in recipient patients with irregular anti-red blood cell antibodies, or in risk situations (such as patients receiving multiple transfusions), it is important to select the red blood cell concentrate unit to be transfused so that the donor's red blood cells are free of the antigens to which the recipient's antibodies are directed or may arise.

[0020] In these patients, tests for blood typing and search and identification of irregular anti-red blood cells are mandatory and are used prophylactically in all recipient patients and in pregnant women before the administration of red blood cell concentrates to detect potential feto-maternal incompatibility. Additional tests by direct compatibility tests with donor red blood cells in the presence of recipient serum or plasma are also mandatory to confirm compatibility. In these cases, neither agglutination nor decomposition reactions are found in the techniques used in IAT.

[0021] The search for so-called irregular antibodies requires the detection of the presence or absence of immunoglobulins against various red blood cell antigens in the blood of an individual. Direct testing or direct Coombs analysis is performed when the antibodies are already fixed in the body. In the case of an alloantibody search, the aim is to reveal the fixation of these immunoglobulins in a red blood test of known antigens by the indirect Coombs technique.

[0022] In the field of immunohematology, there are a large number of methods and devices for detecting and / or identifying red blood cell antigens and for detecting and / or identifying anti-red blood cell antibodies, but they have a number of disadvantages.

[0023] The slide method involves mixing a drop of donor or recipient blood with anti-A (anti-ABO1), anti-B (anti-ABO2) and anti-D (anti-RH1), respectively, on a glass slide or white porcelain support. Agglutination can be visually observed, from which the ABO and Rh D systems of the blood can be determined. The test is completed within 5-10 minutes and is inexpensive. However, it is an insensitive method. It cannot be tested for weakly or very low-reactive antigens, its results are difficult to interpret, and in addition, low titers of anti-A (anti-ABO1) or anti-B (anti-ABO2) can lead to false positive or false negative results. Due to its rapid results, it is very valuable for preliminary blood type matching in emergency situations. However, it is not reliable enough to fully ensure safe blood transfusions.

[0024] The tube test includes the determination of both Beth-Vincent and Simonin-Michon analyses. In this method, for Beth-Vincent grouping (blood typing), blood cells are placed in two test tubes together with saline as a dilution medium, and then one drop of each anti-A (anti-ABO1) and anti-B (anti-ABO2) is added to these samples respectively. These tubes are subjected to centrifugation for several minutes, and then the resulting matrix is ​​gently shaken to observe agglutination. The purpose of centrifugation is to ensure enhanced chemical interactions, especially for weaker antibodies to react, thereby causing agglutination. In a similar manner, the Simonin-Michon reverse grouping (blood typing) can be performed by treating serum with a red blood cell reagent set for A1 and B (and optionally O and A2 blood types), and the subsequent agglutination pattern is monitored. In general, the tube method is much more sensitive than the slide test and requires a low volume of reagents, and some unexpected antigens can also be detected. However, in infants, reverse grouping (blood typing) is somewhat difficult to perform because the amount of antibodies they produce is insufficient and cannot be determined. Since this test is a manual method, its use for high-throughput analysis is very limited.

[0025] In the classical method, the microplate technique includes a large number of small tubes containing a few microliters (μl) of reagents that are processed for the blood sample. After centrifugation and incubation, the subsequent agglutination can be checked by an automatic reading device. This method is a more sensitive and rapid analysis for blood typing and detection / identification of anti-red blood cell antibodies, with low reagent volumes and the feasibility of automation for high-throughput analysis. However, since the microplate technique requires a centrifugation stage, followed by a stirring step, there is a risk of eliminating weak agglutinations in the case of unsuccessful resuspending (resuspending, restoring) strong agglutinations. They must be performed under visual inspection and special attention must be paid to the adhesion of certain reagents.

[0026] The filtration technique by gel test is a standard procedure for quantifying 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 for other blood groups in microtubes under controlled incubation and centrifugation. For reverse blood typing and detection / identification of anti-red blood cell antibodies, plasma or serum is mixed with red blood cells of known antigenicity under controlled incubation and centrifugation. The gel particles capture the agglutinates, while the non-agglutinated blood cells are allowed to pass through the column. The analysis time can be reduced by using glass beads instead of gel material, because faster centrifugation speeds can be achieved in this way, which gives rapid results. This technique is sensitive, simple and relatively easy to operate for less trained personnel. However, the main risk is that some agglutination is not detected, especially during plasma testing for ABO blood typing, because small agglutinates are dissociated by shear forces when entering the gel.

[0027] Moreover, all of these techniques suffer from a major drawback, as they require a centrifugation step to decant the red blood cells or pass them through a gel, a limiting step that adds considerable time and cost to the analysis and requires the use of bulky centrifuges that are difficult to handle.

[0028] Other methods based on molecular imprinting (for review: Mujahid, 2016, Sensors), antibody array technology surfaces based on plasma resonance imaging (Houngkamhang, 2013, sensors; Pipatpanukul, 2018, Biosensors and Bioelectronics), quantum dot-bead analysis (Xu, 2017, International Journal of Nanomedicine), or capillary or vacuum-driven microfluidics (Zhai, 2017, Lab on a Chip), as well as other methods are also described in the related art.

[0029] WO 2012010666 describes a magnetic immunodiagnostic method for antibody / antigen complexes for determining blood group and phenotype

[0030] For example, the immunofiltration method described in application EP2167967 involves capturing the analyte present in the sample when the sample passes through a porous membrane with a capture element and displaying its presence by a display element. This method has significant sensitivity and specificity problems, because when the sample is deposited, the sample diffuses and imbibes the porous membrane, and many analytes are lost in the dead volume of the porous membrane or pass outside the capture area. The same applies to the display solution. Therefore, it is necessary to increase the size of the absorption system to be able to deposit larger volumes of the sample to be tested and the display solution, thereby preventing the test from being miniaturized, its dynamics are controlled, free from receiving display agents and can be used by automatic pipettes.

[0031] In order to try to solve this problem and concentrate the signal emitted after the antigen / antibody reaction, it is proposed in applications CA1312265 or W002052263 to insert a pierced hydrophobic structure above and below the hydrophilic porous membrane to force the flow through the capture point. However, for these devices, there is still the problem of centrifugal diffusion from the point, and the display elements that pass through the capture point and are not fixed thereto will be able to diffuse centrifugally in the hydrophilic porous membrane and be stored at the periphery of the point.

[0032] Another major problem with today's existing immunofiltration devices is controlling the speed of sample movement through the membrane and in some cases the pre-incubation time. These times are important because the interactions between the capture element (usually an antibody) and the analyte (usually an antigen) and between the analyte and the display element have specific kinetics. Without a specific system, the speed of flow through the hydrophilic membrane is very fast (500 μl / min).

[0033] To control the flow, it has been proposed to use a piston, in particular in application US2008318342.

[0034] In order to control the pre-incubation time, it is proposed in application WO03016902 to use a device in two parts; the upper part comprises the sample collection area and the porous membrane, while the lower part comprises the porous membrane and the absorption membrane.

[0035] Such mechanical methods are difficult to perform using automated equipment because they require the development and use of specialized systems. They also expose the professional to any projections during processing.

[0036] Another method described in EP0334015 consists in using an additional membrane below the first membrane to control the flow, but the proposed device does not solve the problems associated with the diffusion of display elements and samples in hydrophilic porous membranes.

[0037] Platelet transfusions (also called platelet concentrates) are used to prevent or treat bleeding in people with low platelet counts or poor platelet function. 9 People with hemorrhages are usually given prophylactic transfusions. In those with hemorrhages, transfusions are typically given at a rate of less than 50 x 10 9 Platelets are supplied by injection into a vein. Platelets can be produced from whole blood or by apheresis. So far, 33 human platelet alloantigens (HPA) have been identified for six functionally important platelet glycoprotein (GP) complexes. The largest number of validated HPAs (20 of 33) are located on the GPIIb / IIIa complex, which acts as a receptor for an important ligand in mediating hemostasis and inflammation. These include HPA-1a, which is the most common HPA in FNAIT and PTP in the Caucasian population. Other platelet GP complexes GPIb / V / IX, GPIa / IIa and CD109 express the remaining 13 HPAs. Among the validated HPAs, 12 appear as six serologically and genetically determined double allele "systems", in which the "a" form represents a higher frequency allele, and the "b" form represents a lower frequency allele. Twenty-one other HPAs are low frequency or rare antigens whose putative higher frequency "a" alleles have not yet been identified as antibody specific. In addition to the HPA markers, platelets express ABO and human leukocyte antigen (HLA) antigens. Blood type matching (ABO, RH1) is typically recommended before donating platelets.

[0038] Accurate typing of patients for HPA antigens is required in several different clinical situations, and blood services need to maintain HPA-typed panels of apheresis platelet and whole blood providers to support HPA-allo-immunized patients. The value of serological HPA phenotypes is limited because typically few platelets can be obtained from thrombocytopenic patients, and reliable serological typing reagents for HPA antigens other than HPA-1a and HPA-5b are rare. In contrast to red blood cell phenotypes, monoclonal antibodies have not been developed for HPA typing other than HPA-1a. However, several rapid screening assays using polyclonal or recombinant anti-HPA-1a have recently been published. These phenotypic assays may complement genotyping assays to provide HPA-selected provider panels.

[0039] It is important to ensure compatibility between the donor of the platelets and the recipient of the platelets. However, due to the unavailability of matched platelets, unmatched platelets are often used. Incompatibility can lead to alloimmune platelet disorders, including fetal and neonatal alloimmune thrombocytopenia (FNAIT), post-transfusion purpura (PTP), and multiple platelet refractoriness (MPR).

[0040] FNAIT, often referred to as fetal-maternal alloimmune thrombocytopenia (FMAIT), results from maternal immunization against fetal platelet alloantigens inherited from the father. Maternal IgG alloantibodies then cross the placenta and cause immune destruction of platelets in utero, a condition similar to hemolytic disease of the newborn.

[0041] Post-transfusion purpura (PTP) is a rare but serious condition that occurs approximately one week after transfusion of any blood product containing platelets or platelet membranes. It is presumed that the patient may be sensitized by a previous pregnancy or transfusion and respond to the secondary challenge of incompatible platelets by producing high titers of HPA (and often HLA) antibodies. The resulting immune destruction of transfused platelets may contribute to the transfusion reactions that commonly occur.

[0042] MPR is defined as an inadequate platelet count following randomized ABO-identical donor platelet transfusion. It is a common complication in patients who receive multiple platelet transfusions.

[0043] Although a variety of techniques for HPA antibody detection have been developed over the past 25 years, four techniques or variations thereof have become the most common: (1) platelet immunofluorescence test (PIFT); (2) monoclonal antibody immobilization of platelet antigens (MAIPA) assay; (3) solid-phase erythrocyte adhesion assay; and (4) various ELISA-based techniques. However, proficiency in antiplatelet antibody detection depends on a variety of factors, including the technique, cell panels, and operator experience, which can make some of these techniques expensive, difficult to perform, and less reliable.

[0044] In order to detect potential transfusion (blood transfusion) transmissible infections, many diagnostic tests that utilize immune responses have been developed during the past four decades. The most commonly used tests are designed to detect antigens of the source of infection, such as viral antigens, bacterial antigens, and parasitic antigens, or antibodies against the source of infection produced by the immune response during the detection of infection. Nevertheless, these tests are not suitable for all situations, and each test has limitations that must be understood and taken into account when selecting. Typically, immunodiagnosis is based on the use of antibodies as reagents.

[0045] Different methods are used in immunodiagnostic testing. Some of these methods are described below.

[0046] Immunoprecipitation is the simplest immunoassay method, which measures the amount of precipitate that forms after reagent antibodies are incubated with the sample and react with their respective antigens to form insoluble aggregates. Immunoprecipitation reactions can be qualitative or quantitative.

[0047] The presence of antibody or specific antigen in particle immunoassay detection sample is tested using particles coated with antigen or appropriate antibody. By connecting several antibodies to particles (gelatin or latex), particles can simultaneously bind many molecules and promote agglutination. This greatly accelerates the speed of visible reaction. Treponema pallidum particle agglutination analysis (also referred to as TPPA test) is another example (Manavi et al. 2006 Int. J. STD AIDS) of particle immunoassay for detecting antibodies against pathogens of syphilis (which may be a transmissible source of infection by blood transfusion). It is a kind of indirect agglutination analysis, in which gelatin particles are sensitized by Treponema pallidum antigen. When patient serum is positive for syphilis, particles aggregate and form agglomerates, and negative test shows that there is no agglomerate of gelatin particles.

[0048] Immunoturbidimetric assays are another example of immunoassays developed to detect infection. When immune complexes formed by the combination of antibody and antigen are too small to precipitate, an instrument called a turbidimeter can be used to measure these complexes because they scatter incident light. Antigen concentration can be determined within a few minutes of the reaction.

[0049] Radioimmunoassay (RIA) uses radioactive isotopes to label antigens or antibodies. The isotope emits gamma rays, which are usually measured after removing the unbound radioactive label. Compared with other immunoassays, the main advantages of RIA are higher sensitivity, easy signal detection, and well-established rapid analysis. The main disadvantages are the health and safety risks of using radiation, and the time and cost associated with maintaining a licensed radiation safety and disposal plan. For this reason, in conventional clinical laboratory practice, RIA has been largely replaced by enzyme (EIA), fluorescence (FIA) or chemiluminescence (CLIA) immunoassays.

[0050] EIA, FIA and CLIA are currently the most commonly used tests for detecting viral, bacterial and parasitic infections, especially transfusion-transmissible infections in blood donor samples. Enzyme-linked immunosorbent assay (ELISA) is one of the most widely used EIA methods for detecting infectious diseases (infectious diseases). There are many patents and publications showing the performance of this analysis for detecting infectious agents.

[0051] These methods are similar in their principle and differ only in the mode of detection of the resulting immune complexes: colorimetric for EIA and ELISA, and fluorescence and photometric measurements resulting from enzymatic / chemical reactions for FIA and CLIA. Fluorescence and chemiluminescence measurements are inherently more sensitive than colorimetry. Therefore, these methods have a higher analytical sensitivity than EIA methods that employ optical density measurements.

[0052] There are also quick / simple unit tests that have no intermediate stages and are disposable, they are used only once and are discarded. Most of these tests are based on immunochromatography, where the sample (blood, plasma or serum) passes through an inert strip and reacts with a reagent (antibody or antigen) that is pre-fixed on the strip. Any positive reaction is visualized as a dot or band that appears on the strip. These tests are easy to use and do not require additional reagents except for the reagents included in the kit. They also give simple qualitative results within a few minutes. However, these tests are not suitable for screening of a large number of samples.

[0053] In order to overcome the above disadvantages, the applicant has implemented an in vitro diagnostic device for detecting at least one reaction between an antigen and an antibody specific for the antigen from a blood sample or one of its components, comprising a hydrophobic porous membrane, wherein the surface of at least one hydrophilic reaction area intended to receive the sample is less important than the surface of the hydrophobic porous membrane. This device is described, for example, in patent application WO2013 / 186482. It compensates for some of the disadvantages of the above tests, in particular of the immunofiltration test, by:

[0054] -Prevents return of display agents due to false positives

[0055] -Increased sensitivity from using smaller volumes

[0056] -Miniaturization and automation of the system

[0057] -Control of reaction kinetics without mechanical manipulation of the device.

[0058] Despite the obvious advantages of the device, when the diagnosis includes the use of a capture agent and when the capture agent is an antibody (for detecting the corresponding antigen in the sample) or an antigen (for detecting the corresponding antibody in the sample), there is still a need to improve the sensitivity and specificity of the diagnosis performed. When using the device, a portion of the capture agent (e.g., an antibody against a blood cell antigen) disposed on the hydrophilic reaction area during the diagnostic test is adsorbed by the porous membrane together with the solution used for antibody dilution. Therefore, the specificity and sensitivity of the test are reduced. In order to compensate for this "loss" of the capture agent, it is necessary to add a large amount of capture agent, which can increase the cost of the diagnostic test.

[0059] In order to overcome this drawback, the inventors of the present invention have tested several technical solutions, such as using thicker porous membranes or less porous membranes, but without significant results.

[0060] In contrast to other possibilities of studying the properties of the porous membrane or other structural elements of the device, the inventors unexpectedly found that the addition of specially selected beads placed on the reaction area of ​​the porous membrane (on the surface of which the capture agent (e.g., antibody or antigen) can be immobilized or adsorbed) allows the capture agent to be retained on the surface of the membrane and thus improves the signal emitted when the capture agent binds to the analyte (e.g., antigen or antibody). Therefore, the use of such beads allows the specificity and sensitivity of the previously disclosed devices to be improved without the need to use large amounts of capture agent.

[0061] Beads with antibodies and / or antigens immobilized on their surface are known in the art for diagnostic purposes. For example, International Application WO 95 / 31731 discloses a method for detecting red blood cell antigens, which comprises adding a sample containing blood cells to a column containing particles on the surface of which specific anti-red blood cell antibodies are immobilized via protein ligands. However, the method described does not use a device comprising a hydrophilized porous membrane and does not address (solve) the problems associated with the absorption of capture agents through the membrane.

[0062] Therefore, the combination of partially hydrophilized porous membranes with beads having antibodies and / or antigens directly or indirectly immobilized or adsorbed on the surface as capture agents for performing in vitro diagnostic methods with high specificity and sensitivity is surprising in view of the state of the relevant prior art. Summary of the invention

[0063] As described above, in order to improve the sensitivity and specificity of detection and / or identification of antigens and / or antibodies and to reduce the amount of analyte used, the inventors implemented a device similar to the device disclosed in patent application WO2013 / 186482, which also includes beads arranged on a reaction area, wherein the antibodies and / or antigens are immobilized or adsorbed on the surface of the beads.

[0064] According to one aspect, the present invention therefore relates to an in vitro diagnostic device for detecting and / or identifying antigens and / or antibodies from a biological fluid sample, preferably from a blood sample or a blood component sample, comprising:

[0065] - a support (support, carrier), and

[0066] - a hydrophobic porous membrane arranged in a support, comprising at least one hydrophilic reaction area intended to receive the sample, the surface of the hydrophilic reaction area being smaller than the surface of the hydrophobic porous membrane, the surface of the hydrophilic reaction area comprising at least one antibody and / or antigen, wherein the antibody and / or antigen is immobilized or adsorbed on the bead surface.

[0067] The inventors of the present invention have conducted several tests and have demonstrated that the in vitro diagnostic device of the present invention can be used to detect and / or identify different types of antigens or antibodies. To this end, different types of antibodies and / or antigens can be fixed or adsorbed on the bead surface. These antibodies can be selected from antibodies against red blood cell antigens, antibodies or anti-immunoglobulins against platelet antigens, antiviral antigens, antibacterial antigens and anti-parasitic antigens. These antigens can be selected from red blood cell antigens, platelet antigens, viral antigens, bacterial antigens and parasitic antigens.

[0068] According to another aspect, the present invention therefore relates to the use of an in vitro device according to the invention for determining and / or identifying:

[0069] - at least one antigen selected from the group consisting of red blood cell antigens, platelet antigens, viral antigens, bacterial antigens and parasite antigens including a mixed-field population;

[0070] - 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-parasite antibodies

[0071] It comes from a biological fluid sample, preferably from a blood sample or a blood component sample.

[0072] The inventors have demonstrated in particular that the in vitro device of the invention can be used in an in vitro method for detecting and / or identifying red blood cell antigens and / or in vivo sensitized red blood cell and / or platelet antigens from a biological fluid sample, in particular from a blood or blood component sample.

[0073] According to another aspect, the present invention therefore relates to an in vitro method for detecting and / or identifying red blood cell antigens and / or in vivo sensitized red blood cell and / or platelet antigens from a blood sample or a blood component sample, comprising the following steps:

[0074] - adding a solution containing the sample onto the reaction area of ​​the in vitro device according to the invention,

[0075] - Reading before washing as sample loading control

[0076] – depositing a cleaning solution onto the reaction area, and

[0077] - if a red or pink spot appears in the reaction area, the presence of the antigen or the blood cell is determined by determining the presence of an antigen / antibody interaction, or if a colorless spot appears in the reaction area, the absence of the antigen or the blood cell is determined by determining the absence of an antigen / antibody interaction.

[0078] Furthermore, the inventors have 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 from a biological fluid sample, in particular from a blood or blood component sample.

[0079] Another aspect of the invention therefore relates to an in vitro method for detecting and or identifying anti-red blood cell antibodies from a blood sample or a blood component sample, comprising the following steps:

[0080] - incubating the sample to be tested with a buffer and a red blood cell test substance of known antigenicity, or incubating the recipient's plasma or serum with the donor's red blood cells,

[0081] - depositing the mixture onto a reaction zone of an in vitro device according to the invention,

[0082] - depositing a cleaning solution onto the reaction area, and

[0083] - The presence of anti-red blood cell antibodies is determined by determining the presence of interacting antibodies / antigens if red or pink spots appear in the reaction area, or the absence of interacting antibodies / antigens is determined by determining their absence if colorless spots appear in the reaction area.

[0084] 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 biological fluid sample, in particular from a blood or blood component sample.

[0085] Another aspect of the present invention therefore relates to an in vitro method for detecting and or identifying anti-platelet antibodies from a blood sample or a blood component sample, comprising the following steps:

[0086] - incubating the sample to be tested with a buffer and a platelet test substance of known antigenicity, or incubating the recipient's plasma or serum with the donor's platelets,

[0087] - depositing the mixture onto a reaction zone of an in vitro device according to the invention,

[0088] - depositing a cleaning solution onto the reaction area, and

[0089] - The presence of antiplatelet antibodies is determined by determining the presence of interacting antibodies / antigens if red or pink spots appear in the reaction area, or the absence of interacting antibodies / antigens is determined by determining their absence if colorless spots appear in the reaction area.

[0090] The in vitro device of the present invention can also be used in an in vitro method for detecting and / or identifying different types of antigens selected from viral antigens, bacterial antigens and parasitic antigens or different types of antibodies selected from anti-viral antibodies, anti-bacterial antibodies and / or anti-parasitic antibodies, which are produced against viral, bacterial or parasitic antigens during infection.

[0091] A further aspect of the present invention therefore relates to an in vitro method for detecting and / or identifying an antigen selected from the group consisting of viral antigens, bacterial antigens and parasite antigens from a biological fluid sample, preferably from a blood sample or a blood component sample, comprising the following steps:

[0092] - optionally incubating the sample to be tested with a buffer,

[0093] - adding a solution containing the sample or a mixture of the sample and a buffer onto the reaction area of ​​the in vitro device according to the invention,

[0094] -optionally taking a reading before washing as a sample loading control;

[0095] - optionally depositing a cleaning solution on the reaction area, and

[0096] - if a colored spot appears in the reaction area, the presence of the antigen is determined by determining the presence of an antigen / antibody interaction, or if a colorless spot appears in the reaction area, the absence of the antigen is determined by determining the absence of an antigen / antibody reaction.

[0097] According to another aspect, the present invention also relates to an in vitro method for detecting and / or identifying antibodies selected from antiviral antibodies, antibacterial antibodies and / or antiparasitic antibodies from a biological fluid sample, preferably from a blood sample or a blood component sample, comprising the following steps:

[0098] - optionally incubating the sample to be tested with a buffer,

[0099] - adding a solution containing the sample or a mixture of the sample and a buffer onto the reaction area of ​​the in vitro device according to the invention,

[0100] -optionally taking a reading before washing as a sample loading control;

[0101] - optionally depositing a cleaning solution on the reaction area, and

[0102] - if a colored spot appears in the reaction area, the presence of the antigen is determined by determining the presence of an antigen / antibody interaction, or if a colorless spot appears in the reaction area, the absence of the antigen is determined by determining the absence of an antigen / antibody reaction.

[0103] The characteristics and advantages of the invention will emerge from the following detailed description, from examples demonstrating some embodiments of the invention also shown in a set of drawings accompanying the present application. DETAILED DESCRIPTION

[0104] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the invention belongs. Some of these definitions are mentioned in the background technology section of this application and are applicable to the present invention. Specific definitions are also given in the detailed description below.

[0105] The extracorporeal device of the present invention

[0106] As described above, the inventors of the present invention have implemented an in vitro device for detecting and / or identifying antigens and / or antibodies with high specificity and sensitivity.

[0107] In one aspect, the in vitro diagnostic device of the present invention is an in vitro diagnostic device for detecting and / or identifying antigens and / or antibodies from a biological fluid sample, preferably from a blood sample or a blood component sample, comprising:

[0108] - a support, and

[0109] - a hydrophobic porous membrane arranged in the support, comprising at least one hydrophilic reaction area intended to receive the sample, the surface of the hydrophilic reaction area being smaller than the surface of the hydrophobic porous membrane, the surface of the hydrophilic reaction area comprising at least one antibody and / or antigen, wherein the antibody and / or antigen is immobilized or adsorbed on the bead surface.

[0110] The device of the present invention is suitable for detecting and / or identifying several types of antigens or antibodies. The detection and / or identification is carried out by an antigen bound to an antibody fixed to an adsorbed (fixed or adsorbed) on the bead surface or by an antibody bound to an antigen fixed or adsorbed on the bead surface. In this context, depending on the type of antigen or antibody to be detected and / or identified, any antibody and / or antigen can be fixed or adsorbed on the bead surface in the device of the present invention. The in vitro device of the present invention is therefore not only used to detect and / or identify specific (specific, specific) antigens or antibodies (such as red blood cell antigens and anti-red blood cell antibodies), but also suitable for detecting and / or identifying different types of antigens and antibodies. By fixing the antigens or antibodies to which the antibodies or antigens of interest are respectively connected on the bead surface, those skilled in the art will be able to adapt the in vitro device of the present invention according to the antibodies or antigens of interest to be detected and / or identified.

[0111] Thus, the term "antibody" is used herein in the broadest sense and specifically encompasses 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 fragments. Antibodies reactive with a specific antigen can be produced by recombinant methods, such as selecting a library of recombinant antibodies in a phage or similar vector, or by immunizing an animal with the antigen or a nucleic acid encoding the antigen.

[0112] Typical antibodies include two identical light chains and two identical heavy chains linked by disulfide bonds. In the meaning of the present invention, the term "light chain" refers to a mammalian immunoglobulin light chain lambda (λ) or kappa (κ) having two consecutive domains (domains), one constant domain and one variable domain.

[0113] The term "heavy chain" refers to the chains of mammalian immunoglobulins denoted as: α, δ, ε, γ and μ. Within these five classes, immunoglobulins can also be distributed in 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 the same in all antibodies of the same isotype. The variable region of each heavy chain consists of a single Ig domain.

[0114] The "variable region" of an antibody refers to the amino-terminal domain of the heavy or light chain of an antibody. The variable domain of the heavy chain may be referred to as "VH." The variable domain of the light chain may be referred to as "VL." These domains are generally the most variable parts of the antibody and contain the antigen binding site. Each variable region contains three segments, called "complementarity determining regions" ("CDRs") or "hypervariable regions," which are primarily responsible for binding to the epitope of the antigen. Thus, the CDRs direct the specificity of antibody binding. They are often referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. The most highly conserved portions of the variable region are called "framework regions."

[0115] In one embodiment of the invention, the antibodies used in the present invention, particularly the antibodies fixed or adsorbed on the surface of the beads, are polyclonal antibodies. "Polyclonal antibodies" are antibodies produced between or in the presence of one or more other different antibodies. Typically, polyclonal antibodies are produced by B lymphocytes in the presence of several other B lymphocytes that produce different antibodies. Typically, polyclonal antibodies are directly obtained from immunized animals.

[0116] According to another embodiment, the antibody used in the present invention, in particular the antibody immobilized or adsorbed on the surface of the beads is a monoclonal antibody.

[0117] As used herein, the term "monoclonal antibody" refers to an antibody derived from an almost homogeneous antibody population. More particularly, the individual antibodies in the population are identical, except for a few possible natural mutations that can be found in minimal proportion. In other words, monoclonal antibodies are composed of a homogeneous antibody population derived from the growth of a single cell clone (e.g., 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, etc.), and are generally characterized by one and only one heavy chain of a class and subclass and only one type of light chain. Monoclonal antibodies are highly specific and are directed against a single antigen.

[0118] As used herein, a "chimeric antibody" is an antibody in which the constant region, or a portion thereof, is altered, replaced or exchanged so that the variable region is linked to a constant region of a different species, or belonging to another antibody class or subclass.

[0119] As used herein, "antigen-binding fragment" is the region of an antibody that binds to an antigen. It consists of one constant domain and one variable domain of each of the heavy and light chains.

[0120] According to a preferred embodiment of the present invention, the antibody is selected from anti-red blood cell antibodies, anti-platelet antibodies, antibodies against viral antigens, antibodies against bacterial antigens and antibodies against parasite antigens, preferably selected from anti-red blood cell antibodies or anti-platelet antibodies.

[0121] As used herein, the term "anti-platelet antibody" or "antibody to platelet antigen" refers to one or more antibodies to one or more platelet antigens. Preferably, the antibody to platelet antigen used in the present invention may be a purified or semi-purified monoclonal antibody, a purified or semi-purified polyclonal antibody or an antiserum.

[0122] As used herein, the term "antibodies against viral antigens" refers to one or more antibodies against viral antigens, which are produced when an individual is infected. Preferably, the antibodies against viral antigens used in the present invention may be purified or semi-purified monoclonal antibodies, purified or semi-purified polyclonal antibodies or antiserum. The antiviral antibodies detected and / or identified by the in vitro device of the present invention may be selected from antibodies that are specific to the following antigens: Chikungunya fever, cytomegalovirus, dengue fever, Ebola, EBV, encephalitis, feline leukemia virus, Hantavirus, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Hepatitis E, Herpes, HIV, HTLV, Influenza Lassa, Measles, Mumps, Norovirus, Papillomavirus Parvovirus, Rubella, SARS, Chickenpox, West Nile Virus and Zika. In particular, for transfusion safety, detection of antibodies against HIV, HTLV Hepatitis B or Hepatitis C is mandatory. In particular, the in vitro device of the present invention is used for detecting and / or identifying HIV, hepatitis B and hepatitis C, more particularly hepatitis B.

[0123] As used herein, the term "antibodies against bacterial antigens" relates to one or more antibodies against bacterial antigens, which are produced when an individual is infected. Preferably, the antibodies against bacterial antigens used in the present invention may be purified or semi-purified monoclonal antibodies, purified or semi-purified polyclonal antibodies or antiserum. These antibodies may be polyclonal or monoclonal antibodies, in particular monoclonal anti-bacterial antibodies well known to those skilled in the art. For example, such antibodies may be selected from Gram-positive or Gram-negative bacteria, in particular from Borrelia, Chlamydia, Helicobacter Pylori, Mycoplasma, S. Typhi, Yersinia, Proteus, Pseudomonas, Escherichia, Klebsiella, Acinetobacter, Clostridium, Serratia, Propionibacterium, Staphylococcus, Bacillus, Treponema and Enterococcus. In particular, the anti-bacterial antibodies are directed against specific antigens of the Treponema genus, in particular Treponema pallidum which causes syphilis.

[0124] As used herein, the term "antibodies against parasite antigens" relates to one or more antibodies against parasite antigens, which are produced when an individual is infected. Preferably, the antibodies against parasite antigens used in the present invention may be purified or semi-purified monoclonal antibodies, purified or semi-purified polyclonal antibodies or antiserum. The anti-parasite antibodies used in the present invention are selected from the genus Toxoplasma, the genus Trypanosoma, and the genus Plasmodium. In particular, the antibodies are against specific antigens of Trypanosoma Cruzi, which causes Chagas' disease. More particularly, the antibodies are against Plasmodium, preferably against Plasmodium facliparum, which causes malaria.

[0125] As used herein, the term "anti-immunoglobulin" relates to antibodies directed against one or more immunoglobulins, such as IgG, IgM, IgA, IgD and IgE.

[0126] As used herein, the term "anti-red blood cell antibody" or "antibody to red blood cell antigen" refers to one or more antibodies against one or more red blood cell antigens. Preferably, the antibody to red blood cell antigen used in the present invention may be a purified or semi-purified monoclonal antibody, a purified or semi-purified polyclonal antibody or an antiserum. Lectins or lectins may also be used.

[0127] The following is a non-exhaustive list of antibodies and antigens used in the present invention:

[0128]

[0129]

[0130] *Polyclonal antibodies

[0131] Table 1 : Non-exhaustive list of red blood cell antigens and antibodies

[0132] Most of the antibodies listed above can be purchased from the applicant Diagast. According to one embodiment of the present invention, the antibody immobilized or adsorbed on the bead surface is an anti-red blood cell antibody selected from any one of the antibodies in Table 1. For example, the anti-red blood cell antibody can be selected from the antibodies Anti-A, Anti-B, Anti-AB, Anti-D, Anti-RH2, Anti-RH3, Anti-RH4, Anti-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.

[0133] In the context of the present invention, the term "antigen" relates to a predetermined molecule to which an antibody can selectively bind. The target antigen may be a polypeptide, a carbohydrate, a nucleic acid, a lipid, a hapten, a glycoprotein or a glycolipid or any other naturally occurring or synthetic compound. Preferably, the target antigen is a polypeptide, a glycoprotein or a glycolipid.

[0134] According to a preferred embodiment of the present invention, the antigen is selected from red blood cell antigens (as listed in Table 1), platelet antigens, viral antigens, bacterial antigens and parasite antigens, more preferably selected from red blood cell antibodies or viral antigens. Preferably, the antigen used in the present invention can be a purified or semi-purified natural antigen, a recombinant or synthetic antigen.

[0135] As used herein, the term "viral antigen" refers to an antigen with multiple antigenicity, which is strain-specific and closely associated with viral particles. The antigen may be a natural or recombinant protein. The viral antigen may be, for example, selected from antigens specific to: Chikungunya fever, cytomegalovirus, dengue fever, Ebola, EBV, encephalitis, feline leukemia virus, Hantavirus, Hepatitis A, Hepatitis B (HBsAg), Hepatitis C (HCsAg), Hepatitis D, Hepatitis E, Herpes, HIV, HTLV, Influenza Lassa, Measles, Mumps, Norovirus, Papillomavirus Parvovirus, Rubella, SARS, Chickenpox, West Nile Virus and Zika. In particular, the present invention relates to the identification and / or detection of antigens HBsAg and HCsAg, because they are mandatory for transfusion safety, more particularly for HBsAg.

[0136] As used herein, the term "bacterial antigen" refers to a molecule found on the surface of a bacterial organism. Bacterial antigens may be selected from any of a bacterial strain, 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, antigens specific for Treponema pallidum, the substance that causes syphilis, may be detected and / or identified, or may be immobilized and / or adsorbed on the bead surface.

[0137] As used herein, the term "parasite antigen" relates to a molecule found on the surface of a parasite cell. The parasite may be as defined above. Preferably, the parasite antigen is selected from antigens specific for Toxoplasma, Trypanosoma, Plasmodium, more preferably for Plasmodium, in particular for Plasmodium falciparum, which causes malaria.

[0138] A non-exhaustive list of antigens and antibodies against viral, bacterial and parasitic antigens is given in Table 2 below:

[0139]

[0140] Table 2: Non-exhaustive list of antigens and antibodies against viral, bacterial and parasitic antigens

[0141] As used herein, the term "platelet antigen" refers to platelet antigens associated with all immunogenic molecules present at the surface of platelets, capable of eliciting the production of antibodies against these molecules and / or allowing their recognition when desired. Platelet antigens can be selected, for example, from the biallelic antigens HPA-1, -2, -3, -4, -5 and -15.

[0142] As used herein, the term "red blood cell antigens" (also known as blood group antigens) refers to all immunogenic molecules present at the surface of red blood cells that are capable of eliciting the production of antibodies against these molecules and / or allowing their recognition when desired. Red blood cell antigens include mixed field population antigens. A non-exhaustive list of red blood cell antigens is shown in Table 1.

[0143] As used herein, the term "mixed field population" refers to two populations of red blood cells with different antigens, which can be observed after a transfusion with compatible red blood cells between the donor and recipient (transfusion of O red blood cells in A or B patient) or after a bone marrow transplant. The in vitro device of the present invention allows the detection of samples with mixed field populations.

[0144] As used herein, the term "in vivo sensitized red blood cells" refers to red blood cells to which immunoglobulins (IgG) or complement (C3d) are bound in vivo to their membrane surfaces. In vivo sensitized red blood cells may occur in different clinical hemolytic conditions, including hemolytic transfusion reactions, hemolytic disease of the fetus and newborn (HDFN), autoimmune hemolytic anemia (AIHA), and drug-induced antibodies in patients.

[0145] According to another embodiment of the present invention, the antibody and / or antigen is directly or indirectly immobilized or adsorbed on the bead surface.

[0146] There are currently several methods for attaching biological ligands to beads used as solid supports in immunological tests and assays, including adsorption to simple (flat) polymer beads, covalent attachment to surface functionalized microspheres. The adsorption mechanism is mainly based on hydrophobic (van der Waals, London-type) attraction between the hydrophobic portion of the adsorbed ligand and the polymer surface of the bead. This is a method that mainly consists of covalent coupling to immobilize biomolecules (when very active and stable bead reagents are required), while indirect immobilization is mediated by anti-immunoglobulins (e.g., anti-human globulin (AHG)) covalently fixed or adsorbed on the beads, which capture a second antibody against the antigen of interest.

[0147] When the antibody and / or antigen is directly immobilized or adsorbed on the bead surface, it is done by contacting the beads with the antibody and / or antigen. For example, the solution containing the antibody and / or antigen may be a non-denaturing buffer comprising a pH solution stable between pH 4 and pH 10, preferably between pH 6.5 and pH 7.8, even more preferably between pH 7 and pH 7.5. The antibody may be covalently linked or adsorbed.

[0148] The antibody solution is selected from the culture supernatant or concentrate of the supernatant, purified antibody, semi-purified or enriched antibody. The antigen solution is selected from purified or semi-purified natural antigen, recombinant or synthetic antigen. Antibodies and / or antigens can be added to adjuvants intended to maintain their microbiological stability (e.g., sodium azide, antibiotics), and adjuvants such as sugars (sucrose, glucose, trehalose) for their conformational stability, and any other reagents known to those skilled in the art for performing these functions.

[0149] According to one embodiment, the bead surface comprises at least one chemical group selected from aldehyde groups, chloromethyl groups, NHS groups and carboxyl groups.

[0150] In particular, when the bead surface contains an aldehyde group, this group reacts with the amino group of the antibody, which allows the direct immobilization of the antibody against the antigen to be determined and / or identified or the antibody for indirect immobilization (as described below) to the surface of the bead. In the case of immobilizing an antigen, this group reacts with the amino group of the antigen, which allows the direct immobilization of the antigen recognized by the antibody to be determined and / or identified.

[0151] In another particular embodiment, the bead surface comprises NHS groups or also known as N-hydroxysuccinimide (NHS) functional groups, preferably it comprises NHS-ester functional groups.

[0152] According to another embodiment of the present invention, antibodies against antigens to be determined and / or identified or against known antigens can be indirectly fixed to the bead surface by another antibody (e.g., anti-immunoglobulin). Such anti-immunoglobulins are preferably directed against immunoglobulins, more preferably IgM or IgG. In this case, if the bead surface contains an aldehyde group, the amino group of the anti-immunoglobulin reacts with the aldehyde group, and the anti-immunoglobulin is fixed to the bead surface in this way. The antibody against the antigen to be determined and / or identified is then fixed to the anti-immunoglobulin by protein / protein interactions.

[0153] According to yet another embodiment, antibodies against the antigen to be determined and / or identified or known antigens can be indirectly immobilized to the bead surface via a ligand selected from proteins having affinity for the antibody (eg protein A, protein G and / or protein L).

[0154] The beads placed on the porous membrane of the in vitro device of the invention can be made of different types of materials, such as glass, polymers, and in particular latex. Preferably, the beads are selected from 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.

[0155] More preferably, the beads are latex beads, especially aldehyde functionalized latex beads.

[0156] According to one embodiment, 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% of the solution deposited on the hydrophilization reaction area of ​​the porous membrane, the deposition volume of the solution being between 1 and 100 μl, preferably between 1 and 50 μl, more preferably between 5 and 50 μl, and even more preferably between 1 and 10 μl or between 5 and 10 μl.

[0157] The size of the beads is selected according to the size of the pores of the hydrophobic porous membrane. The size of the beads must be larger than the size of the pores of the porous membrane to avoid absorption of the beads in the porous membrane. However, in order to avoid agglomeration phenomena, the size of the beads does not have to be very important. The skilled person will be able to select the size of the beads according to the size of the pores of the porous membrane.

[0158] In a preferred embodiment of the present invention, the size of the beads is between 1 μm and 130 μm, preferably between 5 μm and 20 μm and more preferably it is 9 μm.

[0159] According to one embodiment of the extracorporeal device according to the invention, the thickness of the porous membrane is between 0.4 mm and 2 mm, preferably between 0.6 mm and 1.5 mm.

[0160] The hydrophobic porous membrane may comprise any material that is not altered by aqueous solvents. The material may be selected in particular from natural polymers, such as nitrocellulose polymers, cellulose, chemically modified or not, or from synthetic polymers such as polyethylene, high density polyethylene (HDPE) or fluorinated polymers such as polyvinylidene fluoride (PVDF), preferably polyethylene. These polymers may or may not be functionalized with reagent groups capable of producing attachment to a capture agent subsequently used.

[0161] The hydrophobic porous membrane comprises at least one hydrophilic reaction area on which the beads are deposited. The surface of the reaction area is smaller than the surface of the hydrophobic porous membrane, ie the membrane cannot be completely hydrophilized.

[0162] The hydrophilic reaction region(s) of the porous membrane are preferably rendered hydrophilic by the local addition of a surfactant without modification of the chemical functionality of the porous substrate by prior chemical or physical treatment of the hydrophobic porous membrane.

[0163] In the context of the present invention, the term "surfactant" is intended to mean any hydrophilizing agent, ie any substance capable of rendering a hydrophobic membrane hydrophilic in such a way that a sample containing the analyte to be detected can pass therethrough by capillary action.

[0164] The surfactant used can be selected from natural surfactants, natural surfactants obtained by chemical modification or chemical synthesis. Preferably, it is a nonionic surfactant, such as Triton X-100, Tween 20 or saponin, polyoxyethylene or nonyl-β-D-glucoside.

[0165] The surfactant may be diluted in an aqueous solution or an organic solvent (e.g., ethanol) at a concentration between 0.01 and 5% (weight / volume). Preferably, the surfactant used to render the membrane locally hydrophilic is used at a concentration between 0.1% and 3%, more preferably between 0.5 and 2%, and even more preferably between 0.5 and 1% (weight / volume).

[0166] In order to hydrophilize the hydrophobic porous membrane, 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 deposited on the membrane.

[0167] Therefore, according to one embodiment, the hydrophilic reaction area of ​​the hydrophobic porous membrane is rendered hydrophilic by a surfactant, which is preferably used in a concentration of 0.01 to 5% w / v, more preferably 0.1% to 3% w / v, and even more preferably 0.5% to 2% w / v of the solution, and wherein 0.1 to 5 μl, preferably 0.3 to 3 μl and more preferably 0.5 to 2 μl of the solution containing the surfactant is placed on the hydrophobic porous membrane.

[0168] The concentration of the surfactant used, which is related to the other characteristics of the membrane, in particular the porosity and thickness, controls the speed of movement of the fluid through the membrane. It is generally believed that in order to make a membrane intended to receive the capture element hydrophilic, it is not necessary to exceed a maximum dosage of 0.1% for Triton X-100 and 0.05% for Tween. However, due to the special characteristics of the membrane according to the invention, detergents capable of making the membrane locally hydrophilic can be used up to 5%, preferably up to 3%, especially for Triton X-100 or Tween-20, without destroying the reactivity of this area, which makes the hydrophilization of the membrane easier.

[0169] The same hydrophobic membrane may comprise several hydrophilic reactive regions, provided that these regions do not intersect.

[0170] The reaction zone may be of all geometrical forms, but is preferably in the form of a circle or a spot with a diameter between 0.3 mm and 20 mm.

[0171] The reaction zone may be hydrophilic throughout the thickness of the porous membrane and / or at the surface.

[0172] The reaction areas may have a single degree of hydrophilization, ie the reaction areas have the same degree of hydrophilization.

[0173] According to one embodiment, the reaction zone may include several regions with different hydrophilicity. For example, the reaction zone may include two hydrophilic regions, wherein the hydrophilicity at the center of the reaction zone is higher than that at the periphery. These hydrophilic regions are preferably only at the surface of the membrane.

[0174] The reaction zone may also include two hydrophilic regions of different degrees of hydrophilization, one at the surface and the other in the thickness.

[0175] In case the reaction zone comprises two hydrophilic regions, the reaction zone has been rendered hydrophilic using two different surfactants without modifying the chemical functionality of the porous substrate by prior chemical or physical treatment of the porous membrane.

[0176] Advantageously, when detecting a mixed field population from a blood or blood component sample, a configuration having two reaction zones with different hydrophilic regions, particularly at the surface, is useful. This configuration is also particularly suitable for detecting and / or identifying specific antibodies and / or antigens in the sample to be tested.

[0177] According to one embodiment, the in vitro device of the invention may comprise an absorbent layer (or also referred to as absorbent membrane) arranged below the porous membrane. This layer absorbs liquid deposited at the level of the reaction zone, which liquid is not retained by the porous membrane, in particular when the reaction zone is hydrophilic throughout the thickness of the membrane.

[0178] The absorbent layer may include a material capable of passive absorption by capillary action, such as absorbent paper, cellulose, etc., or may be made of an absorbent polymer. By way of example, the following products may be cited as the absorbent layer:

[0179] - C048, C068, C083, C248(Merck)

[0180] - CF3, CF4, CF10, Grade 470, CF5, CF6, CF7, Grade 900, Grade 300

[0181] - Grades 601, 642, 631, 238, 237, 222, 243, 320 (Munktell)

[0182] -Pall Grades 111, 113, 133, 165, 197, 8975, 8964, 8301 (Pall Corporation), membranes for direct collection, storage, and efficient rapid release of biological samples for clinical diagnosis Ultra

[0183] -Cleanis Super absorbent pads.

[0184] -Mc Arlaid T-499, SCP-300-TCF, T-089, T-183-5 and SCP-200-TCF

[0185] The composition of the absorbent membrane and its dimensions must be chosen so that it can absorb all the solutions (V in μl) used during the test. 全部 Each membrane is characterized by its absorbency (in μl / cm 2 C), select the membrane and its size (in cm 2 The D) represented by the equation: D>V 全部 / C.

[0186] According to one embodiment, the extracorporeal device of the present invention further comprises at least one further layer, which is arranged between the porous hydrophobic membrane and the adsorbent layer, and which is made of a drainage material. The drainage material is, for example, cotton wool, NA7150PES, NT9610HY, NT9750HY NV170F, NV250F, NV340F, (Subrenat) or Packtex HY050B.

[0187] The porous membrane and optionally the absorbent layer and the drainage layer are arranged in a support of the device.

[0188] The support of the in vitro device according to the invention is preferably a rigid support. It may for example be a housing.

[0189] The support preferably comprises a rigid material that does not allow the liquid to escape. It may in particular be a plastic material, such as polypropylene, polyethylene, polystyrene, acrylonitrile-butadiene-styrene, polyethylene terephthalate, polycarbonate, polyamide, polyvinyl chloride, polymethyl methacrylate.

[0190] The support of the extracorporeal device of the present invention preferably comprises two parts, a lower part on which the hydrophobic porous membrane, the drainage layer and the absorbent layer are disposed, and an upper part covering the lower part.

[0191] Preferably, an absorbent layer is arranged on the surface of the lower part of the support, on which a hydrophobic porous membrane is arranged. More preferably, a drainage layer is arranged between the absorbent layer and the porous membrane. Even more preferably, a foam layer is arranged on the surface of the lower part of the support, on which the absorbent layer is deposited, then the drainage layer, on which a hydrophobic porous membrane including at least one hydrophilic reaction area is deposited, the surface of the hydrophilic reaction area being smaller than the surface of the membrane, and on which beads are deposited, the beads having antibodies ( Figure 1 a and b).

[0192] The upper portion of the support covers the lower portion, such as a hydrophobic porous membrane, and any or all of the other layers, when they are present in the support, may be encapsulated in the support.

[0193] The upper part of the support preferably comprises at least one or more openings. The opening (one or more) corresponds to the collection area of ​​the sample deposited on the hydrophilic reaction area. The hydrophilic area can have the same size as the bottom of the opening, smaller or larger (of the same size), provided that the two hydrophilic areas are always separated by the hydrophobic area on the membrane.

[0194] The collection area must be sized such that it can contain at least the maximum volume of the reaction mixture or sample to be tested deposited on the reaction membrane.

[0195] According to one embodiment, if the different parts of the extracorporeal device of the invention are supplied as a kit, they can be assembled by the user.The device can be supplied in one piece.

[0196] As mentioned above, the in vitro devices of the present invention can also detect and / or identify antibodies.

[0197] According to one embodiment, the detection of antibodies is performed by binding to specific known antigens that are fixed and / or adsorbed on the surface of the beads. The antibodies to be identified and / or determined in the biological fluid sample can specifically bind to the known antigens. The detection of this binding allows the detection of antibodies that are specifically directed against the antigen.

[0198] According to another embodiment of the present invention, the detection of antibodies can be carried out by fixing or adsorbing anti-immunoglobulins (antibodies) on the bead surface, wherein the anti-immunoglobulins are anti-immunoglobulins that are capable of binding to other antibodies (to be detected and / or identified) present in the sample to be tested. The second antibody is then detected and / or identified by using a display agent (e.g., a labeled antigen that specifically binds to the antibody).

[0199] According to another embodiment of the present invention, the detection of the antibody can be carried out by fixing or adsorbing a protein with affinity for the antibody on the bead surface, the protein being capable of binding to the antibody (to be detected and / or identified) present in the sample to be tested. The antibody is then detected and / or identified by using a display agent (e.g., a labeled antigen that specifically binds to the antibody).

[0200] In one embodiment, when determining and / or identifying anti-red blood cell antibodies, the beads are immobilized or adsorbed with anti-immunoglobulins (anti-IgG and / or anti-IgM), which will capture circulating anti-red blood cell antibodies in a blood sample, especially serum or plasma. The display and identification of these antibodies can be performed using red blood cells carrying the corresponding antigens.

[0201] In one embodiment, when measuring and / or identifying anti-red blood cell antibodies, beads are immobilized or adsorbed with anti-immunoglobulins (anti-IgG and / or anti-IgM), which will capture anti-red blood cell antibodies present on the surface of red blood cells sensitized in vivo. The display and identification of these antibodies is performed by the red blood cells sensitized in vivo themselves.

[0202] In one embodiment, beads are immobilized or adsorbed with anti-C3d antibodies, which capture complement-sensitized red blood cells in vivo, resulting from complement activation by in vivo fixation of IgM anti-red blood cell antibodies. The display and identification of these antibodies is performed by the in vivo sensitized red blood cells themselves.

[0203] The same principle can be applied to antibodies against viral, bacterial or parasitic antigens that circulate in the blood of the tested individual suspected of being infected, using, for example, a different display system, such as a second set of colored beads coupled to the corresponding antigen or antibody, which is conjugated to an enzyme (e.g. horseradish peroxidase) or to colloidal gold nanoparticles.

[0204] The in vitro device of the present invention is intended for the determination and / or identification of antigens and / or antibodies from a biological fluid sample.

[0205] In the context of the present invention, the term "biological fluid sample" relates to any sample obtained from a bioorganic fluid produced by a living organism. The biological fluid is selected from the group consisting of extracellular fluid, intravascular fluid, interstitial fluid, lymph and transcellular fluid. In particular, the biological fluid sample is selected from the group consisting of blood and blood components, urine, saliva, etc.

[0206] In a more preferred embodiment, the biological fluid sample is a blood sample or a blood component sample. "Blood sample" or "blood component sample" means total blood or one of its components, in particular selected from the red blood cell fraction, the white blood cell fraction, platelets, plasma or serum.

[0207] In particular, when the in vitro device of the invention is used for the detection and / or identification of red blood cell antigens and / or antibodies against these antigens, the sample is a blood sample or a blood component sample.

[0208] In particular, when the in vitro device of the invention is used for the detection and / or identification of viral, bacterial or parasitic antigens and / or antibodies against these antigens, the sample is a blood sample or a blood component sample.

[0209] The sample as described above may be obtained from any organism, in particular from a mammal and more particularly from a human.

[0210] Preferred embodiments

[0211] Various preferred specific features corresponding to various general elements of the extracorporeal device according to the present invention have been described above in the parts specifically related to the elements. In the context of the present invention, each list of suitable features for a specific element and each specific feature disclosed for a specific element can be combined with any other general element, the list of suitable features for the other elements or any specific feature disclosed for the other elements.

[0212] In particular, preferred embodiments of an element of the extracorporeal device according to the invention may be combined with any other general element or with preferred embodiments of said other elements.

[0213] Preferred embodiments correspond to those in which at least one element is limited to the preferred embodiments, as listed in Table 3 below:

[0214]

[0215]

[0216] Table 3.Preferred embodiments of the various elements of the extracorporeal device according to the invention

[0217] Purpose (use) of the extracorporeal device of the present invention

[0218] The in vitro devices of the present invention can be used to determine and / or identify antigens and / or antibodies from a biological fluid sample.

[0219] As described above, the device is suitable for detecting and / or identifying any antigen and / or any antibody, depending on the antibody and / or antigen immobilized or adsorbed on the bead surface.

[0220] The inventors have conducted studies to demonstrate that the in vitro device of the present invention is suitable for use with antibodies immobilized on the surface of beads having structural differences, such as antibodies against blood cell antigens and antibodies against at least one antigen specific to a virus, bacteria or parasite. For example, the antibodies tested are antibodies against HCV (HCsAg) or HBV (HBsAg) antigens, which cause hepatitis C and B, respectively, and the antibodies can be fixed or adsorbed on the surface of the beads to detect and / or identify the listed antigens, respectively. Preferably, the antibodies are antibodies against the antigen HBV (HBsAg.)

[0221] In particular, the inventors have demonstrated that the in vitro device of the present invention can be used to detect and / or identify antigens and / or antibodies from red blood cells, viruses, bacteria and parasites from biological fluids, in particular from blood samples or blood component samples.

[0222] According to another aspect, the present invention therefore relates to the use of an in vitro device according to the invention for determining and / or identifying:

[0223] - at least one antigen selected from the group consisting of red blood cell antigens, platelet antigens, viral antigens, bacterial antigens and parasite antigens including a mixed-field population;

[0224] - at least one antibody selected from the group consisting of anti-red blood cell antibodies, anti-platelet antigens, anti-viral antibodies, anti-bacterial antibodies and anti-parasite antibodies from a biological fluid sample, preferably from a blood sample or a blood component sample

[0225] It comes from a biological fluid sample, preferably from a blood sample or a blood component sample.

[0226] As described above, depending on the type of antibody or antigen immobilized or adsorbed on the bead surface, the in vitro device of the present invention can detect and / or identify different types of antigens or antibodies.

[0227] In particular, the in vitro device of the present invention is used to detect and / or identify antigens of the group comprising red blood cell antigens, platelet antigens, viral antigens, bacterial antigens and parasite antigens. More particularly, the device is used to detect and / or identify red blood cell antigens.

[0228] The in vitro device of the present invention can be used in particular for determining and / or identifying (or also phenotyping) red blood cells, i.e. determining antigens at their surface. Phenotyping of red blood cells generally means determining and / or identifying the blood group. In the context of the present invention, the term "blood group" relates to a blood group selected, for example, from the ABO, D, Rh, Kell, MNS, JK (Kidd), FY (Duffy), LE (Lewis), LU (Lutheran), P1PK systems.

[0229] The in vitro device of the present invention is also used to detect and / or identify antibodies selected from anti-red blood cell antibodies or also referred to as anti-blood cell antibodies, including autoantibodies, alloantibodies, xenoantibodies and cold antiglobulins. In particular, these antibodies are detected and / or identified from blood samples or blood component samples.

[0230] The extracorporeal device of the present invention may be used, inter alia, for:

[0231] - Simonin-Michon test, which identifies the presence of anti–A (anti-ABO1) or anti–B (anti-ABO2) antibodies;

[0232] - Search or identify antibodies against cellular antigens, especially red blood cells, in terms of searching for alloantibodies, autoantibodies, xenoantibodies or even cold agglutinins by indirect antiglobulin testing;

[0233] -Searching for compatibility between the donor's red blood cells and the recipient's blood by performing a cross-matching reaction;

[0234] -A direct antiglobulin test is used to search for antibodies that are fixed to red blood cells in the body.

[0235] As used herein, the term "allogeneic antibody" refers to one or more antibodies produced by an individual that react with one or more antigens from a genetically different individual of the same species. These allogeneic antibodies are produced by immunization after a blood transfusion, during pregnancy, after a transplantation or graft.

[0236] As used herein, the term "autoantibodies" relates to one or more antibodies produced by an individual against one or more antigens of the same individual. The detection of autoantibodies is preferably performed after a blood transfusion or in case of hemolytic anemia (autoimmune, drug-induced or after a blood transfusion).

[0237] As used herein, the term "xenoantibodies" relates to one or more antibodies produced by an individual against one or more antigens of another species.

[0238] As used herein, the term "cold agglutinin" relates to antibodies that cause agglutination of red blood cells at low temperatures.

[0239] As used herein, the term "cross-match reaction" refers to a test performed prior to a transfusion to determine whether the donor's red blood cells are compatible with the blood or plasma of the intended recipient.

[0240] The antiviral, antibacterial and antiparasitic antibodies detected and / or identified by in vitro means are immune antibodies produced in response to viral, bacterial or parasitic antigens when an individual is infected.

[0241] The in vitro devices of the present invention may also be used to detect the presence of pathogens by assaying for the presence of one or more viral, bacterial or parasitic antigens.

[0242] The method of the present invention

[0243] The in vitro device of the present invention is particularly suitable for use in an in vitro method for detecting and / or identifying antigens selected from red blood cell antigens including mixed field populations, in vivo sensitized red blood cells (also known as C3d-positive cells), platelet antigens, viral antigens, bacterial antigens, parasite antigens, or for detecting and / or identifying anti-red blood cell antibodies, including in vivo sensitized red blood cells (IgG-positive cells), and / or anti-platelet antibodies and / or antibodies against viral, bacterial and parasite antigens, which are produced against viral, bacterial or parasite antigens when an individual is infected and are present in the blood.

[0244] According to another aspect, the present invention thus relates to an in vitro method for detecting and / or identifying red blood cell antigens from a blood sample or a blood component sample comprising a mixed field population, and / or in vivo sensitized red blood cells (C3d-positive cells) and / or platelet antigens, comprising the following steps:

[0245] - adding a solution containing the sample onto the reaction area of ​​the in vitro device according to the invention, and

[0246] - Read before washing as sample loading control;

[0247] - depositing a cleaning solution onto the reaction area, and

[0248] - If a red or pink spot appears in the reaction area, the presence of the antigen or blood cells is determined by determining the presence of an antigen / antibody interaction, or if a colorless spot appears in the reaction area, the absence of the antigen or blood cells is determined by determining the absence of an antigen / antibody interaction.

[0249] According to one embodiment, before the sample to be tested is deposited on the reaction area including the beads, the hydration of the reaction area that has been hydrophilized can be performed with the aid of a buffer solution. The buffer may include a stable pH 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 osmotic pressure between 250mOsm and 800mOsm, preferably between 300mOsm and 600mOsm. The solution may optionally contain a low concentration of a surfactant (0.01 to 0.05% m / v Tween 20), a saturating agent (BSA) and / or an agent capable of making the antigen-antibody reaction potential. The buffer may optionally have protease activity, for example obtained by adding enzymes such as papain or bromelain. The buffer may optionally contain a polycationic agent such as polybrene or polylysine to make the reaction potential and keep the red blood cells in contact with the capture element for a longer time.

[0250] Thus, the above in vitro method optionally comprises a step of hydrating the porous membrane prior to adding the sample to be tested.

[0251] According to another embodiment, the above in vitro method further comprises another optional step of diluting the red blood cells to be phenotyped in a buffer solution, in particular a buffer solution known to favor immunohematological reactions, the buffer solution optionally containing additives, such as a buffer comprising a stable pH 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 osmotic pressure between 250mOsm and 800mOsm, preferably between 300mOsm and 600mOsm. The solution may optionally contain a low concentration of a detergent (especially 0.01 to 0.05% m / v Tween 20), a saturant (e.g. BSA) and / or an agent capable of making the antigen-antibody reaction potent. The buffer may optionally have protease activity, for example obtained by adding enzymes such as papain or bromelain. The buffer may optionally contain a polycationic agent such as polybrene or polylysine to make the reaction potent and keep the ball in contact with the capture element for a longer time.

[0252] According to another embodiment, the above-mentioned method of the present invention optionally includes an additional step, namely, before depositing the tested sample on the reaction area, incubating the tested sample at a temperature preferably between 15 and 40°C, in particular between 18°C ​​and 37°C, for a time of 2sec to 30min and in particular 1min to 20min.

[0253] Similarly, for reading the result, a buffer wash step must be performed. The wash buffer preferably comprises PBS, TBS or a saline solution with a pH between 2 and 10, preferably between 5 and 9. The osmotic pressure of the buffer must be controlled to avoid hemolysis (haemolysis, dissolution) of red blood cells. The buffer must be selected so as not to disengage the display agent or colored analyte that is directly or indirectly fixed to the capture agent (the antibody fixed and / or adsorbed on the bead surface). Surprisingly, for use according to the in vitro device of the present invention, a slightly hypertonic wash solution (i.e., between 300mOsm and 800mOsm) obtained by the presence of a salt reagent (e.g., NaCl) or a nonionic osmotic agent (e.g., glycine or taurine) is preferably used. The buffer can optionally be colored with a color that contrasts with the color of the display agent. For example, if the display agent is a red blood cell, the wash buffer solution can be dyed blue or green. A low dose of a surfactant can also be added to the wash solution to eliminate background noise. These surfactants are preferably nonionic surfactants and in particular esters of sugars, especially polyoxyethylene esters of sorbitan (Tween).

[0254] During use of the in vitro device, the liquid can be deposited, in particular, with the aid of a pipette system or a capillary replication system.

[0255] In the in vitro method, the hydrophilization of the reaction area of ​​the porous membrane of the in vitro device according to the invention may be a thickness hydrophilization, ie a hydrophilization performed over the entire thickness of the porous membrane.

[0256] The hydrophilization can be carried out with the aid of an aqueous solution of Triton X100 having a concentration between 0.01% m / v and 5% m / v, preferably between 0.1% m / v and 3% w / v, preferably between 0.5% m / v and 2% m / v, in a volume between 0.1 μl and 5 μl, preferably between 0.3 μl and 3 μl and more preferably between 0.5 μl and 2 μl.

[0257] In some applications, when using Triton, the hydrophilization of the porous membrane may be partial, that is, not performed over the entire thickness of the membrane.

[0258] If the red blood cells present in the sample carry an antigen recognized by the specific (particular) antibody fixed or adsorbed on the bead surface (antigen of the red blood cells or antibody fixed on the red blood cells sensitized in vivo), the red blood cells remain fixed on the bead surface provided on the reaction area despite washing, and the reaction area remains red or pink. If the red blood cells present in the sample do not carry an antigen recognized by the antibody fixed or adsorbed on the bead surface, the red blood cells are washed by the washing solution, and the reaction area remains colorless.

[0259] Reading can be visual or automatic.

[0260] The detection and / or identification of sensitized red blood cells in vivo allows the detection of autoantibodies or alloantibodies adsorbed at the surface of red blood cells. In particular, the test is based on the use of beads coupled to anti-immunoglobulin (anti-IgG) or anti-C3d antibodies (for IgM), which capture the red blood cells to be tested via the IgG or C3d immobilized on their membranes (the formation of which is induced by IgM).

[0261] Advantageously, the device according to the invention requires neither centrifugation nor agitation nor vacuuming nor ad hoc devices. It can also be used completely autonomously manually and can be easily automated on a robot.

[0262] Furthermore, the inventors have 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 agglutinins) from a biological fluid sample, in particular from a blood or blood component sample.

[0263] Another aspect of the present invention therefore relates to an in vitro method for detecting and / or identifying anti-red blood cell antibodies from a blood sample or a blood component sample, comprising the following steps:

[0264] - incubating the sample to be tested with a buffer and a red blood cell test substance of known phenotype,

[0265] - depositing the mixture onto a reaction zone of an in vitro device according to the invention,

[0266] - depositing a cleaning solution onto the reaction area, and

[0267] - The presence of anti-red blood cell antibodies is determined by determining the presence of an antibody / antigen reaction if a red or pink spot appears in the reaction area, or the absence of said antibodies is determined by determining the absence of an antibody / antigen reaction if a colorless spot appears in the reaction area.

[0268] The incubation step may be carried out in the presence of an agent capable of aggregating red blood cells at a temperature ranging from 4 to 40° C. for a period of 3 to 60 min, preferably 5 to 30 min. The skilled person will be able to determine the temperature according to the antibody to be detected and / or identified.

[0269] The buffer used in the incubation step is a low ionic force buffer, such as LISS buffer (eg containing less than 50 mM NaCl).

[0270] According to one embodiment of the method, an agent capable of causing red blood cells to aggregate is added, for example a solution of hexadimethrine bromide may be added to the mixture obtained in the incubation step, preferably at a concentration of between 0.01 and 2% (m / v), even more preferably between 0.05 and 0.5% in the solution. Hexadimethrine bromide promotes red blood cell aggregation.

[0271] According to another embodiment of the method for detecting and / or identifying anti-red blood cell antibodies, human antiglobulins or anti-complement reagents may be deposited on the reaction area after depositing the reagent capable of causing red blood cell aggregation.

[0272] The cleaning solution used to clean the reaction area can be, for example, a hypertonic saline solution having a stable pH 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 an osmotic pressure between 300 mOsm and 800 mOsm. The solution can optionally contain a low concentration of a detergent (e.g., 0.01 to 0.05% m / v Tween 20) and a dye of a color that contrasts with red (blue or green).

[0273] The sample to be deposited may be plasma, serum or whole blood or blood components, diluted or undiluted in a buffer comprising a stable pH solution 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 osmotic pressure between 250mOsm and 800mOsm, preferably between 300mOsm and 600mOsm. The solution may optionally contain a low concentration of a detergent (e.g. 0.01 to 0.05% m / v Tween 20), a saturant (especially BSA) and / or an agent capable of making an antigen-antibody reaction potent. The salinity of the buffer is preferably less than 50mM NaCl.

[0274] If the plasma being tested contains antibodies to the antigens (red blood cell test substances) present in the display reagent, red (or pink) spots will appear in the reaction area. A colorless center means that there is no or undetectable amount of antibodies to the antigens (red blood cell test substances) present in the display reagent.

[0275] Reading can be visual or automatic.

[0276] Another aspect of the present invention further relates to an in vitro method for detecting and or identifying anti-platelet antibodies from a blood sample or a blood component sample, comprising the following steps:

[0277] - incubating the sample to be tested with a buffer and a platelet test substance of known antigenicity, or incubating the recipient's plasma or serum with the donor's platelets,

[0278] - depositing the mixture onto a reaction zone of an in vitro device according to the invention,

[0279] - depositing a cleaning solution onto the reaction area, and

[0280] - The presence of antiplatelet antibodies is determined by determining the presence of antibody / antigen interaction if a red or pink spot appears in the reaction area, or the absence of said antibodies is determined by determining the absence of antigen / antibody interaction if a colorless spot appears in the reaction area.

[0281] The in vitro device of the present invention may also be used in an in vitro method for detecting and / or identifying different types of antigens selected from viral antigens, bacterial antigens and parasitic antigens.

[0282] A further aspect of the present invention therefore relates to an in vitro method for detecting and / or identifying an antigen selected from the group consisting of viral antigens, bacterial antigens and parasite antigens from a biological fluid sample, preferably from a blood sample or a blood component sample, comprising the following steps:

[0283] - optionally incubating the sample to be tested with a buffer,

[0284] - adding a solution containing the sample or a mixture of the sample and a buffer onto the reaction area of ​​the in vitro device according to the invention,

[0285] -optionally taking a reading before washing as a sample loading control;

[0286] - optionally depositing a cleaning solution on the reaction area, and

[0287] - preferably determining the presence of the antigen by determining the presence of an antigen / antibody interaction if a colored spot appears in the reaction area, or preferably determining the absence of the antigen by determining the absence of an antigen / antibody reaction if a colorless spot appears in the reaction area.

[0288] The incubation step may be carried out at a temperature of 4 to 40°C for a period of 3 to 60 min, preferably 5 to 30 min, in the presence of an agent capable of increasing (enhancing) the reaction. The skilled person will be able to determine the appropriate temperature depending on the antibody or antigen to be detected and / or identified.

[0289] The buffer used in the incubation step is a buffer having molecules for increasing the reaction.

[0290] The sample to be deposited may be a biological fluid, examples being: plasma, serum or whole blood or blood components, diluted or undiluted in a buffer comprising a stable pH solution 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 osmotic pressure between 250 mOsm and 800 mOsm, preferably between 300 mOsm and 600 mOsm. The solution may optionally contain a low concentration of a detergent (e.g. 0.01 to 0.05% m / v Tween 20), a saturant (especially BSA) and / or an agent capable of making an antigen-antibody reaction potent. The salinity of the buffer is preferably less than 50 mM, calculated as NaCl.

[0291] The cleaning solution used to clean the reaction area can be, for example, a hypertonic saline solution having a stable pH 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 an osmotic pressure between 300 mOsm and 800 mOsm. The solution can optionally contain a low concentration of a detergent (e.g., 0.01 to 0.05% m / v Tween 20).

[0292] Visualization of antigen / antibody interactions can preferably be performed by using colored beads or HRP (horseradish peroxidase) coupled to antibodies or other molecules that will interact with the corresponding pathogen antigen.

[0293] The skilled person will be able to identify other labeling means that allow detection and / or identification of the antigen.

[0294] If the plasma being tested contains antigens against the antibodies present in the display agent, a colorless spot will appear in the reaction area. The colorless center means that there is no or undetectable amount of antigen against the antibodies present in the display agent.

[0295] Reading can be visual or automatic.

[0296] As mentioned above, it is also possible to use the in vitro device of the present invention to detect and / or identify antibodies to an antigen selected from a virus, bacterium or parasite antigen. In fact, in most cases, for example, when measuring the presence or absence of a pathogen (virus, bacterium or parasite) during a blood transfusion, it is carried out by measuring and / or identifying the antibody to the antigen produced by the infected individual. These antibodies are also referred to in the application as immune antibodies produced for a virus, bacterium or parasite antigen when an individual is infected. They can circulate in the blood of an infected individual.

[0297] Thus, according to a further aspect, the in vitro device of the invention may also be used in an in vitro method for detecting and / or identifying different antibodies against viral antigens, bacterial antigens and parasitic antigens produced upon infection of an individual.

[0298] A further aspect of the invention therefore relates to an in vitro method for detecting and / or identifying antibodies selected from antibodies against viral antigens, bacterial antigens and parasitic antigens from a biological fluid sample, preferably from a blood sample or a blood component sample, preferably said antibodies being produced upon infection, comprising the following steps:

[0299] - Optionally incubate the sample to be tested with a buffer

[0300] - adding a solution containing the sample or a mixture of the sample and a buffer onto the reaction area of ​​the in vitro device according to the invention,

[0301] -optionally taking a reading before washing as a sample loading control;

[0302] - optionally depositing a cleaning solution on the reaction area, and

[0303] - preferably determining the presence of the antibody by determining the presence of an antigen / antibody interaction if a colored spot appears in the reaction area, or preferably determining the absence of the antibody by determining the absence of an antigen / antibody reaction if a colorless spot appears in the reaction area.

[0304] The incubation step may be carried out at a temperature of 4 to 40°C for a period of 3 to 60 min, preferably 5 to 30 min, in the presence of an agent capable of increasing the reaction. The skilled person will be able to determine the appropriate temperature depending on the antibody or antigen to be detected and / or identified.

[0305] The buffer used in the incubation step is a buffer having molecules for increasing the reaction.

[0306] The cleaning solution used to clean the reaction area can be, for example, a hypertonic saline solution having a stable pH 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 an osmotic pressure between 300 mOsm and 800 mOsm. The solution can optionally contain a low concentration of a detergent (e.g., 0.01 to 0.05% m / v Tween 20).

[0307] The sample to be deposited may be a biological fluid, examples being: plasma, serum or whole blood or blood components, diluted or undiluted in a buffer comprising a stable pH solution 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 osmotic pressure between 250 mOsm and 800 mOsm, preferably between 300 mOsm and 600 mOsm. The solution may optionally contain a low concentration of a detergent (e.g. 0.01 to 0.05% m / v Tween 20), a saturant (especially BSA) and / or an agent capable of making an antigen-antibody reaction potent. The salinity of the buffer is preferably less than 50 mM, calculated as NaCl.

[0308] If the plasma being tested contains antibodies against the antigen present in the display agent, a colorless spot will appear in the reaction area. A colorless center means that antibodies against the antigen present in the display agent are absent or undetectable.

[0309] Visualization of the antibody / antigen reaction is preferably performed by using colored beads or HRP (horseradish peroxidase) coupled to the antibody or a molecule that interacts with the corresponding pathogen antigen.

[0310] The skilled artisan will be able to identify other labeling means that allow detection and / or identification of the antibody.

[0311] Reading can be visual or automatic. BRIEF DESCRIPTION OF THE DRAWINGS

[0312] Figure 1 : a. A diagram of a particular embodiment of an in vitro device according to the invention observed in perspective, the device comprising a porous membrane comprising a reaction area on which a plurality of beads of 9 μm in size are deposited, on the surface of which antibodies or antigens are directly or indirectly immobilized or adsorbed. Three layers are shown below the membrane, one layer of drainage material, one layer of absorption material and one layer of foam. Figure 1Also shown is a scanning electron microscope (SEM) image of a portion of a porous membrane comprising beads and antibodies immobilized on its surface. b. Schematic diagram of a particular embodiment of an in vitro device of the present invention, the device comprising a support on which a porous membrane is placed, the porous membrane comprising a hydrophilized reaction area on which a plurality of beads are deposited and on which antibodies are immobilized. Below the porous membrane is shown a Figure 1 The same layers as shown in a.

[0313] Figure 2 :According to WO2013 / 186482( Figure 2 a) and the in vitro device of the present invention ( Figure 2 b) Comparative test for detecting red blood cell antigens using Anti-ABO01, Anti-ABO02, Anti-ABO03, Anti-RH1, Anti-RH2, Anti-RH3, Anti-RH4, Anti-RH5, Anti-KEL1 and negative control.

[0314] Figure 3 : Detection of red blood cell ABO01, ABO02 and ABO3 antigens by IgM antibodies indirectly immobilized on latex beads via proteins on Protein A / G / L.

[0315] Figure 4 : a and b: Detection of red blood cell ABO01 antigen by IgM antibody bound to AHG (anti-human globulin) coupled to chloromethyl latex beads, wherein two different volumes of antibody-coupled beads were loaded on the membrane (a: 20 μl and b: 10 μl). c and d: Detection of red blood cell ABO01 antigen by IgG antibody bound to chloromethyl latex beads coupled to AHG, wherein two different volumes of antibody-coupled beads were loaded on the membrane (c: 20 μl and d: 10 μl). .

[0316] Figure 5 : Detection of red blood cell antigens using IgM-specific antibodies bound to AHG-conjugated epoxide beads. (a: RH2; b: RH3; c: RH4; d: RH5; e: KEL1 antigen and f: negative control)

[0317] Figure 6 : FY1 and RH1 red blood cell antigens were detected by IgG-specific antibodies bound to NHS agarose beads coupled to AHG (anti-human globulin) under the conditions described in Example 3. ab and c. represent different detection procedures, while d. represents different reaction set-ups and detection procedures.

[0318] Figure 7: Detection of red blood cell ABO02 antigen and mixed field populations with varying amounts (1 to 5%) of anti-ABO02 coupled to aldehyde 9 μm latex beads supported on a porous membrane.

[0319] Figure 8 : a. Detection of ABO01, RH2, RH5 and KEL1 red blood cell antigens by different amounts of specific antibodies (0.125 to 4%) coupled to aldehyde 9 μm latex beads on a device comprising a 1.5 mm thick membrane. b. Detection of ABO01, RH2 and KEL11 red blood cell antigens by specific antibodies (4%) coupled to aldehyde 9 μm latex beads on a device comprising a 0.6 mm thick membrane.

[0320] Fig. 9 : Detection of red blood cell ABO1 antigen by specific antibodies coupled to aldehyde 9 μm latex beads supported on an in vitro device comprising a 0.6 mm thick membrane hydrophilized with a. Triton X-100 b. polyoxyethylene and c. nonyl β D-glucoside.

[0321] Fig.10 : a. Detection of red blood cell RH2 antigen by specific antibodies coupled to aldehyde 9 μm latex beads on a device comprising T-499 (left) or SCP-300-TCF (right) as absorbent. b. Detection of red blood cell RH2 antigen by specific antibodies coupled to aldehyde / sulfate 9 μm latex beads on a device comprising T-099 (left) or T-499 (right) as absorbent. c. Detection of red blood cell RH2 antigen by specific antibodies coupled to aldehyde / sulfate 9 μm latex beads on a device comprising T-499 (left) or T-183-5 (right) as absorbent. d. Detection of red blood cell RH2 antigen by specific antibodies coupled to aldehyde / sulfate 9 μm latex beads on a device comprising T-499 (left) or SCP-200-TCF (right) as absorbent. For each image, the device is shown in a disassembled state; from top to bottom: membrane and absorbent on plastic device.

[0322] Fig.11: a. Detection of red blood cell ABO02 antigen by anti-ABO03 antibody coupled to aldehyde 9 μm latex beads on a device comprising cotton wool as a drainage pad. b. Detection of red blood cell ABO02 antigen by anti-ABO03 antibody coupled to aldehyde / sulfate 9 μm latex beads on a device comprising NT 9750HY as a drainage pad. c. Detection of red blood cell ABO02 antigen by anti-ABO03 antibody coupled to aldehyde / sulfate 9 μm latex beads on a device comprising NT 9610HY as a drainage pad. For each image, the device is shown in a disassembled state; from top to bottom: membrane, drainage pad, and absorbent.

[0323] Fig.12 : a. and c. Detection of red blood cell ABO01 or ABO02 antigens by IgM antibodies (anti-ABO01 and anti-ABO02) coupled to aldehyde / sulfate 9 μm latex beads. b. and d. Signal interpretation allowing discrimination of mixed field populations.

[0324] Fig.13 : a. Detection of weak RH1 antigens: Different types of weak RH1 red blood cells (Df1, Df2...) are captured by anti-RH1 (HM16 and ESD1 clones) fixed to A / G / L proteins coupled to aldehyde / sulfate 9μm latex beads. b. Detection of antigens of Duffy (FY1 and FY2), KIDD (JK1 and JK2) and MSN (MNS3 and MNS4 systems): Positive red blood cells are captured by specific antibodies fixed to A / G / L proteins coupled to aldehyde / sulfate 9μm latex beads.

[0325] Fig.14 : Reverse blood typing, test red blood cells of known phenotype (A1, A2, B and O) are incubated with plasma and captured by anti-human IgM (CP6D4 clone) coupled to aldehyde / sulfate 9 μm latex beads.

[0326] Fig.15 : Direct coombs test: a. IgG sensitized red blood cells were captured by anti-human globulin (SA6532, CPC5-1 and 188 33 clones) coupled to aldehyde / sulfate 9 μm latex beads. b. Positive red blood cells were captured by anti-human globulin (SA6532) and / or anti-human C3d (C7610) coupled to aldehyde / sulfate 9 μm latex beads. c. Positive red blood cells were fixed to anti-human globulin (SA6532) and / or anti-human C3d (C7610) and captured on A / G / L proteins coupled to aldehyde / sulfate 9 μm latex beads.

[0327] Fig.16: Detection of antibodies specific for the Plasmodium falciparum MSP1 antigen. a. Using the device of the present invention with HRP detection method b. Using the device of the present invention with 1 μm red beads as detection method c. Using the device of the present invention with colloidal gold nanoparticles as detection method. For each example, negative and positive reactions and quantification of signal intensity are shown (n=3-5).

[0328] Fig.17 Antibodies specific for the Treponema pallidum p15, p17 and p47 antigens were detected using the device of the invention with 1 μm red beads as the detection method. Negative and positive reactions and quantification of signal intensity are shown (n=3).

[0329] Fig.18 Antibodies specific for the HBsAg antigen from the hepatitis B virus were detected using the device of the invention with 1 μm red beads as the detection method. Negative and positive reactions are shown as well as quantification of the signal intensity (n=3).

[0330] Fig.19 . Detection of HBsAg antigen from hepatitis B virus. a. Using the device of the present invention with HRP (horseradish peroxidase) as the detection method (graphs and images). b. Using the device of the present invention with 1 μm red beads as the detection method. For each example, negative and positive reactions and quantification of signal intensity are shown (n=3).

[0331] Example

[0332] Example 1: By coupling anti-red blood cell antibodies directly and indirectly via anti-immunoglobulins IgM and IgG Bead-based in vitro device for red blood cell typing and phenotyping

[0333] A protocol for preparing an in vitro device comprising anti-red blood cell antibodies spotted directly on a membrane on the surface of a reaction zone.

[0334] 1.1. Materials used to prepare in vitro devices:

[0335] - membrane "POREX" 0.6 mm hydrophobic with a porosity of 9 to 12 μm; cotton;

[0336] -Cleanis;

[0337] - Hydrophilization buffer (1% triton, 1% green dye, phosphate buffered saline).

[0338] - Washing buffer (TLA composition);

[0339] - Dilution buffer (Chromasolcoombs);

[0340] - Antibodies (Anti-ABO01, Anti-ABO02, Anti-ABO03, Anti-RH1, Anti-RH2, Anti-RH3, Anti-RH4, Anti-RH5, Anti-KEL1, negative control).

[0341] 1.2. Preparation mode of reaction medium:

[0342] - Deposit 1 μl of hydrophilization solution in each well;

[0343] - Drying at 37°C and 10% humidity for 16 hours;

[0344] - Deposit 2 μl of antibody per well;

[0345] - Dry at 37°C and 10% humidity for 72 hours.

[0346] The materials used to carry out the test are reaction support (carrier), washing buffer and dilution buffer

[0347] 1.3. Test implementation plan:

[0348] - Dilute the red blood cell pellet to 20% in dilution buffer;

[0349] - Deposit 20 μl of the suspension in the wells;

[0350] - Incubate at room temperature for 3 minutes;

[0351] - Deposit 80 μl of wash buffer;

[0352] - Incubate at room temperature for 1 min;

[0353] - Deposit 80 μl of wash buffer.

[0354] 2. Scheme (process) for preparing in vitro devices by coupling anti-red blood cell antibodies to beads directly and indirectly via anti-immunoglobulins IgM and IgG.

[0355] 2.1. Protocol for direct coupling of anti-ABO01 (anti-A), anti-ABO02 (anti-B) and anti-ABO03 (anti-AB) antibodies to latex beads.

[0356] On 1 ml of homogenized 4% beads (Thermo Fisher latex aldehyde / sulfate beads 9 μm, 4% w / v), add 100 μl of 20X phosphate buffered saline and dissolve 2 ml of concentrated antibody. Make up the volume to 4 ml with deionized water. Mix the solution end over end overnight to ensure coupling.

[0357] Excess active groups were capped by suspending 150 μl of 1 M ethanolamine in the bead antibody solution, followed by mixing at room temperature for 2 hours. The coupled beads were then washed thoroughly twice with 3 ml of storage buffer (centrifugation at 2500 g for 5 min before and after washing). Finally, the bead pellet was resuspended in storage buffer to restore the weight of the introduced bead solution.

[0358] 2.2. Indirect conjugation scheme for anti-RH1, anti-RH2, anti-RH3, anti-RH4, anti-RH5 and anti-KEL1 antibodies

[0359] On 1 ml of homogenized 4% beads, add 200 μl of 20X phosphate buffered saline and dissolve 250 μl of 1 mg / ml 6D4 (anti-human IgM, Diagast clone) or 100 μl of 3.69 mg / ml C5-1 (anti-human IgG, Diagast clone). Make up the volume to 4 ml with deionized water. Mix the solution end over end overnight to ensure coupling.

[0360] Excess active groups were capped by suspending 150 μl of 1M ethanolamine in the bead antibody solution, followed by mixing at room temperature for 2 hours. The anti-human globulin (AHG)-coupled beads were then washed thoroughly twice with 3 ml of storage buffer (centrifuged at 2500 g for 5 min before and after washing). The bead pellet was resuspended with 2 ml of concentrated antibody plus 2 ml of phosphate buffered saline. The solution was mixed end-to-end overnight to ensure coupling.

[0361] The resulting beads were then washed extensively twice with 3 ml of storage buffer (centrifugation at 2500 g for 5 min before and after washing). Finally, the bead pellet was resuspended in storage buffer to restore the weight of the introduced bead solution. Negative control beads were directly saturated with ethanolamine.

[0362] The antibody clones used in the Examples are shown in Table 4 below:

[0363] Clone Name Antigen specificity Same type type Coupling type 9113D10 ABO01 IgM Mouse direct 9621A8 ABO02 IgM Mouse direct 152D12 ABO03 IgM Mouse direct P3X255 13G8 RH2 IgM people Indirectly via 6D4 RH3 906 RH3 IgM people Indirectly via 6D4 RH4951 RH4 IgM people Indirectly via 6D4 P3GD512 RH5 IgM people Indirectly via 6D4 K601 KEL1 IgG1 people Indirectly via C51 P3X212 11F1 RH1 IgG1 people Indirectly via C51 6D4 AHG IgG1 Mouse direct C51 AHG IgG1 Mouse direct

[0364] Table 4 : Antibody clones used in the examples.

[0365] The antibody clones in Table 4 are clones produced and sold by the applicant (Diagast, France).

[0366] 2.3. Preparation of reaction device and testing procedures

[0367] The device is a molded plastic box in which a Porex HDPE hydrophobic membrane 0.6 mm thick and with a porosity between 9 and 12 μm, a drainage pad (NA7150 PES from Subrenat) and an absorbent pad (Cleanis) are assembled.

[0368] The porous membrane was hydrophilized 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 at 37°C, 10% humidity for 1 hour. 5 μl of 4% beads coupled to antibodies or negative controls were then spotted and the device was dried at 37°C, 10% humidity for 1 hour before immediate use or long-term storage.

[0369] The assay was initiated by diluting red blood cells at 5% in Chromasolcoombs (Diagast preparation) and introducing 10 μl in each well followed by two 30 μl washes (phosphate buffered saline supplemented with 0.2% Tween 20 and 0.54% green dye).

[0370] In this example, the applicant also compared an in vitro device for blood typing and phenotyping prepared by the above protocol containing anti-red blood cell antibodies coupled to beads with an in vitro device in which the anti-red blood cell antibodies are directly spotted on a porous membrane (similar to the device described in WO2013 / 186482).

[0371] 3. Results

[0372] The comparison results obtained are shown in Figure 2 , where the presence of dark grey spots (corresponding to the red spots in the coloured version) indicates the formation of immune complexes and is therefore a positive reaction (presence of antigen at the red blood cell surface), whereas the presence of colourless spots reflects a negative reaction (absence of antigen at the red blood cell surface).

[0373] from Figure 2 It can be seen that the blood group antibodies anti-ABO01, anti-ABO02, anti-ABO03, anti-RH1, anti RH2, anti-RH3 anti-RH4 anti RH5 and anti-KEL1 directly or indirectly immobilized on the surface of the beads bind in a specific manner to the corresponding antigens present on the surface of the red blood cells of the sample tested. In fact, the surface of the spots where the beads were deposited still retains a good red color, which indicates that the antibodies to the red blood cells are not adsorbed by the porous membrane, which allows specific and sensitive detection of red blood cell antigens ( Figure 2 b).

[0374] from Figure 2a It can be seen that in the in vitro setup without beads, the sensitivity of the reaction with some antibodies, especially Anti-RH1, Anti-RH3, Anti-RH4, Anti-RH5 and Anti-KEL1 antibodies, is low (the intensity of dark grey is very low).

[0375] When compared with the in vitro device of the present invention Figure 2 b The results were comparable to those obtained with the in vitro device without beads. Figure 2 When looking at the results of a, it seems that for antibodies Anti-RH1, Anti-RH3, Anti-RH4, Anti-RH5, and Anti-KEL1KEL1, the gray spots (pots) are Figure 2 a than Figure 2 This is more visible in a, which confirms that the in vitro device of the present invention allows to obtain a more sensitive detection of most anti-red blood cell antigens.

[0376] Example 2: Indirect coupling of red blood cell antibodies via antibody affinity proteins

[0377] 1. Indirect Antibody Conjugation

[0378] Onto 1 ml of homogenized 4% latex beads (Thermo Fisher Latex aldehyde / sulfate 9 μm 4% w / v), 5 or 2.5 μg of protein AGL was dissolved in 3 ml PBS 1X and added. The solution was mixed end-over-end overnight to ensure coupling.

[0379] On 1 ml of centrifuged 4% beads (Thermo Fisher latex aldehyde / sulfate 9 μm 4% w / v) coupled to protein AGL, 3 ml of 1X phosphate buffered saline was added and 1 ml of Anti-ABO01 antibody (91 13D10 Diagast clone) or Anti-ABO02 antibody (96 21A8 Diagast clone) or Anti-ABO03 (152D12 Diagast clone) was dissolved. The solution was mixed end-to-end overnight to ensure coupling.

[0380] The coupled beads were then centrifuged at 2500 g for 5 min. Finally, the bead pellet was resuspended in storage buffer (phosphate buffered saline supplemented with 0.5% bovine serum albumin and 0.25 M sucrose) to restore the weight of the introduced bead solution.

[0381] 2. Preparation of the reaction device and testing procedures

[0382] The device is a molded plastic box in which a Porex HDPE hydrophobic membrane 0.6 mm thick and with a porosity between 9 and 12 μm, a drainage pad (9610HY) and an absorbent pad (Cleanis) are assembled.

[0383] The porous membrane was hydrophilized with 0.5 μl of surfactant solution (phosphate buffered saline supplemented with 2% Triton X-100 and 1% green dye). Then 2 μl of 2% antibody-coupled beads were spotted. Before use, the device was dried at 37°C and 20% humidity for 15 min.

[0384] The detection procedure was started by introducing into each well 50 μl of red blood cells diluted at 1.5% in PBS 1X supplemented with 0.00625% Tween20.

[0385] The first image was acquired before washing. The reaction area was then washed with 30 μl of phosphate buffered saline supplemented with 0.2% Tween-20. The image was acquired after washing.

[0386] 3. Results

[0387] Figure 3 The resulting image is shown, on which the presence of dark grey spots (corresponding to red spots in the colour image) is observed, indicating the formation of immune complexes and therefore a positive reaction, whereas the presence of colourless spots reflects a negative reaction.

[0388] Example 3: Antibodies to red blood cells with different chemical functional groups and There are couplings on beads of different sizes.

[0389] 1. Chloromethyl

[0390] 1.2. Antibody conjugation

[0391] 1.5 ml of phosphate buffer was added to 500 μl of homogenized chloromethyl beads (Thermo Fisher chloromethyl latex beads 4% w / v, 2.0 μm), and the resulting solution was centrifuged at 3000 g for 20 min. The supernatant was discarded, and the beads were then resuspended with a mixture of 950 μl of phosphate buffered saline and 50 μl of concentrated CPC51 or CP6D4 (Diagast AHG). The antibody beads solution was incubated overnight at room temperature on a tube rocker.

[0392] After AHG chemical coupling, the bead solution was centrifuged at 3000g for 15min, and the supernatant was discarded. The beads were washed with 1.5ml of phosphate buffered saline, and the solution was centrifuged at 3000g for 10min. The supernatant was discarded, and the excess active groups were blocked by adding 1ml of 1M ethanolamine, and then mixed at room temperature for 20min. The bead solution coupled with AHG was centrifuged at 3000g for 20min, and then washed with 1.5ml of phosphate buffered saline. 1ml of concentrated anti-A (Anti-ABO1) antibody (clone IgM 91 13 D10 or IgG 162 47 (3) E6, both Diagast clones) was dissolved in the bead particles, and the solution was mixed at room temperature for two hours, then centrifuged and washed with 1.5ml of phosphate buffered saline. Finally, the bead particles were resuspended in a storage buffer to restore the weight of the introduced bead solution.

[0393] 1.2. Preparation of reaction apparatus and testing procedures

[0394] The apparatus is a moulded plastic box in which a Porex HDPE hydrophobic membrane 0.6 mm thick and with a porosity between 9 and 12 μm, a drainage pad (cotton batt from Alan & co) and an absorbent pad (Cleanis) are assembled.

[0395] The porous membrane was hydrophilized with 2.5 μl of surfactant solution (filtered water supplemented with 1% Triton X-100) and then spotted with 20 μl of ( Figure 4 A and 4C) or 10 μl ( Figure 4 B and 4D) 4% antibody-coupled beads. The device was allowed to stand at room temperature for 30 min before use.

[0396] The detection procedure was started by introducing into each well 13 μl of a 10% mixture of 30 μl of red blood cells in chromasolcoombs (Diagast preparation) and 10 μl of a solution of hedimethoxaline bromide.

[0397] The reaction area was washed twice with 25 μl of phosphate buffered saline supplemented with 0.1% Tween-20.

[0398] Results

[0399] Figure 4 The resulting image is shown, on which the presence of dark grey spots (corresponding to red spots in the colour image) is observed, indicating the formation of immune complexes (type A red blood cells) and therefore a positive reaction, whereas the presence of colourless spots reflects a negative reaction (type B red blood cells).

[0400] 2. Epoxy group

[0401] 2.1. Antibody conjugation

[0402] 0.5 g of epoxy resin (Biorad Profinity epoxy resin, supplied as dry powder, particle size 45-90 microns) was weighed out and swelled in 50 ml of phosphate buffered saline with gentle stirring for 30 min. The beads swelled to 5 ml to give approximately 10% solution.

[0403] The beads were centrifuged at 2500 g for 5 min, the supernatant discarded, and the pellet resuspended in 4.5 ml of phosphate buffered saline and centrifuged again.

[0404] 360 μl of concentrated anti IgM AHG (CP6D4 Diagast) was dissolved in 12 ml of phosphate buffered saline and the mixture was added on the bead particles for coupling. The antibody bead solution was incubated on a rotary shaker at room temperature for 1 h 30.

[0405] After AHG chemical coupling, the bead solution was centrifuged at 2500 g for 5 min and the supernatant was discarded. The beads were washed with 4.5 ml of phosphate buffered saline and the solution was centrifuged at 2500 g for 5 min. The supernatant was discarded and excess active groups were capped by adding 3 ml of 1 M ethanolamine and then mixed at room temperature for 45 min. The AHG-coupled bead solution was centrifuged at 2500 g for 5 min and then washed with 4.5 ml of phosphate buffered saline.

[0406] 300 μl of anti-RH2, anti-RH3, anti-RH4 or anti-RH5 (P3X25513G8 Diagast clone, RH3 906 Diagast clone, MS33 Millipore clone and P3GD C512 Diagast clone, respectively) were diluted in 2200 μl of phosphate buffered saline and added to the beads coupled to AHG. For anti-Kell antibody, 1500 μl was diluted in 1000 μl of phosphate buffered saline, and for negative control, in 2500 μl of phosphate buffered saline. The obtained solution was mixed at room temperature for 45 min, then centrifuged and washed. Finally, the bead pellet was resuspended in phosphate buffered saline supplemented with a preservative to recover a 50% solution of the coupled beads.

[0407] 2.2. Preparation of reaction device and testing procedures

[0408] The apparatus is a moulded plastic box in which a Porex HDPE hydrophobic membrane 0.6 mm thick and with a porosity between 9 and 12 μm, a drainage pad (cotton batt from Alan & co) and an absorbent pad (Cleanis) are assembled.

[0409] The porous membrane was hydrophilized with 1 μl of a surfactant solution (filtered water supplemented with 1% Triton X-100) and dried overnight at room temperature. Then 10 μl of 50% antibody-coupled beads were applied. Before use, the device was allowed to stand at room temperature for 30 min.

[0410] The detection procedure was initiated by introducing 10 μl of undiluted red blood cells into each well. The reaction area was washed twice with 40 μl of phosphate buffered saline supplemented with 0.2% Tween-20 and preservatives.

[0411] Results

[0412] Figure 5 The resulting image is shown, on which the presence of dark grey spots (corresponding to the red spots in the colour image) is observed, indicating the formation of immune complexes and therefore a positive reaction, whereas the presence of colourless spots reflects a negative reaction. The negative control yields colourless spots regardless of the red blood cell phenotype.

[0413] 3. N-hydroxysuccinimide (NHS) group

[0414] 3.1. Antibody conjugation

[0415] 1 ml of NHS beads (Pierce TM NHS-Activated Agarose Slurry) was diluted with 5 ml of phosphate buffered saline and centrifuged at 2500 g for 5 min, the supernatant discarded, and the pellet resuspended in 4.5 ml of phosphate buffered saline for 3 additional washes.

[0416] 2 ml of concentrated anti IgG AHG (C51 Diagast) was dissolved in 3 ml of phosphate buffered saline and the mixture was added on the bead particles for coupling. The antibody bead solution was incubated on a rotary shaker at room temperature for one and a half hours.

[0417] After AHG chemical coupling, the bead solution was centrifuged at 2500 g for 5 min and the supernatant was discarded. The beads were washed with 4.5 ml of phosphate buffered saline and the solution was centrifuged at 2500 g for 5 min. The supernatant was discarded and excess active groups were capped by adding 3 ml of 1 M ethanolamine and then mixed at room temperature for 45 min. The AHG-coupled bead solution was centrifuged at 2500 g for 5 min and then washed with 4.5 ml of phosphate buffered saline.

[0418] 10 ml of anti-RH1 and anti-FY1 antibodies (HM16 and F655 Diagast clones, respectively) were added to the AHG-coupled beads. The resulting solution was mixed for 1 hour at room temperature, then centrifuged and washed. Finally, the bead pellet was resuspended in phosphate-buffered saline supplemented with a preservative to obtain a 50% solution of coupled beads.

[0419] 3.2. Preparation of reaction device and testing procedures

[0420] Figure 6 The devices shown above (A, B and C) are molded plastic boxes in which a Porex HDPE hydrophobic membrane 0.6 mm thick and with a porosity between 9 and 12 μm, a drainage pad (cotton batt from Alan & co) and an absorbent pad (Cleanis) are assembled.

[0421] The porous membrane was hydrophilized with 2.5 μl of a surfactant solution (filtered water supplemented with 1% Triton X-100) and dried overnight at 37°C. Then 20 μl of 50% antibody-coupled beads were spotted. The device was allowed to stand at room temperature for 30 min before use.

[0422] Figure 6 A: The detection procedure was started by introducing into each well 7 μl of red blood cells diluted 25% in 0.25% hedimethoxam solution. The reaction area was washed with 20 μl + 30 μl of phosphate buffered saline supplemented with 0.01% Tween-20.

[0423] Figure 6 B: The detection procedure was started by introducing into each well 5 μl of red blood cells diluted 25% in a 0.3% solution of hedimethicone bromide. The reaction area was washed with 20 μl + 30 μl of phosphate buffered saline supplemented with 0.01% Tween-20.

[0424] Figure 6 C: The detection procedure was started by introducing into each well 5 μl of red blood cells diluted 25% in a 0.4% solution of hydroxymethylbenzyl chloride. The reaction area was washed with 20 μl + 30 μl of phosphate buffered saline supplemented with 0.01% Tween-20.

[0425] Figure 6 The device shown on D is a molded plastic box in which a Lydall UHMWPE hydrophobic membrane (Solupor 70M01A) 105 μm thick and 12 μm porosity, a drainage pad (cotton wool from Alan & co) and an absorbent pad (Cleanis) are assembled.

[0426] The porous membrane was hydrophilized with 2 μl of a surfactant solution (filtered water supplemented with 1% Triton X-100) and dried at 37°C for 1 hr 30 min. Then 20 μl of 50% antibody-coupled beads were applied. Before use, the device was allowed to stand at room temperature for 30 min.

[0427] The detection procedure was initiated by introducing into each well 7 μl of red blood cells diluted 25% in a 0.25% solution of hedimethicone bromide. The reaction area was washed with 30 μl + 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.

[0428] Results

[0429] From the image obtained Figure 6 The presence of dark grey spots (corresponding to the red spots in the colour image) is observed on the ELISA, indicating the formation of immune complexes and therefore a positive reaction (detection of RH1 or FY1 antigen), whereas the presence of colourless spots reflects a negative reaction.

[0430] Example 4: Bead Concentration

[0431] The purpose of this exercise was to test different concentrations of beads to be deposited on the porous membrane.

[0432] 1. Antibody Conjugation

[0433] On 1 ml of homogenized 4% beads (Thermo Fisher latex aldehyde / sulfate 9 μm 4% w / v), add 100 μl of 20X phosphate buffered saline and dissolve 2 ml of affinity purified anti-ABO02 antibody (96 21A8 Diagast clone). Make up the volume to 4 ml with deionized water. Mix the solution end-to-end overnight to ensure coupling.

[0434] Excess active groups were capped by suspending 150 μl of 1 M ethanolamine in the bead antibody solution and mixed at room temperature for 2 hours. The coupled beads were then washed thoroughly twice with 3 ml of storage buffer (centrifuged at 2500 g for 5 min before and after washing). Finally, the bead pellet was resuspended in storage buffer to restore the weight of the introduced bead solution.

[0435] 2. Preparation of the reaction device and testing procedures

[0436] The device is a molded plastic box in which a Porex HDPE hydrophobic membrane 0.6 mm thick and with a porosity between 9 and 12 μm, a drainage pad (cotton batt from Alan & co) and an absorbent pad (Cleanis) are assembled.

[0437] The porous membrane was hydrophilized with 1 μl of a surfactant solution (phosphate buffered saline supplemented with 1% Triton X-100 and 1% green dye). Then 10 μl of beads coupled to antibodies at concentrations between 1 and 5% were spotted. Before use, the device was dried at 37°C and 20% humidity for 15 min.

[0438] The detection procedure was started by introducing into each well 50 μl of red blood cells diluted at 0.15% in Chromasolcoombs supplemented with 0.00625% Tween 20. The reaction area was washed once or twice with 30 μl of phosphate buffered saline supplemented with 0.2% Tween-20.

[0439] 3. Results

[0440] Figure 7 The resulting image is shown on which the presence of dark grey spots (corresponding to the red spots in the colour image) is observed, indicating the formation of immune complexes and therefore a positive reaction (ABO02 type red blood cells), whereas the presence of colourless spots reflects a negative reaction. After one or two washes, specificity and sensitivity were respected for bead concentrations from 1 to 5%. Mixed field blood cells can be detected by a decrease in intensity compared to the signal of a 100% positive population.

[0441] Example 5: Thickness of the hydrophobic porous membrane

[0442] 1. Antibody Conjugation

[0443] 1.1. Anti-ABO01 conjugation

[0444] On 1 ml of homogenized 4% beads (Thermofisher latex aldehyde / sulfate 9 μm 4% w / v), add 100 μl of 20X phosphate buffer and dissolve 2 ml of affinity purified anti-ABO01 (2521B8 Diagast clone) antibody. Make up the volume to 4 ml with deionized water. Mix the solution end-to-end overnight to ensure coupling.

[0445] Excess active groups were capped by suspending 150 μl of 1 M ethanolamine in the bead antibody solution and mixed at room temperature for 2 hours. The coupled beads were then washed thoroughly twice with 3 ml of storage buffer (centrifugation at 2500 g for 5 min before and after washing). Finally, the bead pellet was resuspended in storage buffer (phosphate buffered saline supplemented with 0.5% bovine serum albumin and 0.25 M sucrose) to restore the weight of the introduced bead solution.

[0446] 1.2. Anti-RH2, anti-RH5 and anti-KEL1 conjugation

[0447] On 1 ml of homogenized 4% beads, add 200 μl of PBS 20X and dissolve 250 μl of 6D4 (anti-human IgM) at 1 mg / ml for anti-RH2 and anti-RH5 (P3x255 13G8 and P3GD C512 Diagast clones, respectively) or 100 μl of C5-1 (anti-human IgG) at 3.5 mg / ml for anti-KEL1 (601 Diagast clone). Make up the volume to 4 ml with deionized water. Mix the solution end-to-end overnight to ensure coupling.

[0448] Excess active groups were capped by suspending 150 μl of 1 M ethanolamine in the bead antibody solution and mixed at room temperature for 2 hours. The AHG-coupled beads were then washed thoroughly twice with 3 ml of storage buffer (centrifugation at 2500 g for 5 min before and after washing). The bead pellet was resuspended with 2 ml of concentrated antibody plus 2 ml of phosphate buffer. The solution was mixed end-to-end overnight to ensure coupling.

[0449] The coupled beads were then washed twice with 3 ml of storage buffer (centrifugation at 2500 g for 5 min before and after washing). Finally, the bead pellet was resuspended in storage buffer (phosphate buffered saline supplemented with 0.5% bovine serum albumin and 0.25 M sucrose) to restore the weight of the introduced bead solution.

[0450] 1.3. Preparation of reaction apparatus and testing procedures

[0451] Figure 8The device shown on a is a molded plastic box in which a Porex HDPE hydrophobic membrane 1.5 mm thick and with a porosity between 7 and 12 μm, a drainage pad (cotton batt from Alan & co) and an absorbent pad (Cleanis) are assembled.

[0452] The porous membrane was hydrophilized with 1 μl of a surfactant solution (phosphate buffered saline supplemented with 3% Triton X-100, 0.26 M sucrose and 1% green dye). The device was first dried at 37°C, 10% humidity for 1 hour. Then 5 μl of beads coupled to antibodies at concentrations between 0.125 and 4% were spotted. Before use, the device was second dried at 37°C, 10% humidity for 1 hour.

[0453] The detection procedure was initiated by introducing into each well 10 μl of red blood cells diluted 5% in physiological water. The reaction area was washed with 100 μl of phosphate buffered saline supplemented with 0.1% Tween-20 and 0.007% Triton X-100.

[0454] Figure 8 The device shown on b is a molded plastic box in which a Porex HDPE hydrophobic membrane 0.6 mm thick and with a porosity between 9 and 12 μm, a drainage pad (NA7300PES from Subrenat) and an absorbent pad (Cleanis) are assembled.

[0455] The porous membrane was hydrophilized with 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). The device was first dried at 37°C, 10% humidity for 1 hour. Then 5 μl of 4% antibody-coupled beads were applied. Before use, the device was second dried at 37°C, 10% humidity for 1 hour.

[0456] The detection procedure was initiated by introducing into each well 10 μl of red blood cells diluted 5% in physiological water. The reaction area was washed with 100 μl of phosphate buffered saline supplemented with 0.1% Tween-20 and 0.007% Triton X-100.

[0457] Results

[0458] Figure 8 The presence of dark grey spots (red spots) on a indicates the formation of immune complexes and is therefore a positive reaction. The sensitivity is observed with decreasing intensity for bead concentrations from 4 to 0.125%.

[0459] Figure 8The presence of dark grey spots (red spots) on b indicates the formation of immune complexes and is therefore a positive reaction, whereas the presence of colourless spots reflects a negative reaction.

[0460] Example 6: Type and concentration of surfactant used to hydrophilize porous membranes

[0461] 1. Anti-ABO01 conjugation

[0462] On 1 ml of homogenized 4% beads (Thermo Fisher latex aldehyde / sulfate 9 μm 4% w / v), add 100 μl of 20X phosphate buffered saline and dissolve 1 ml of affinity anti-ABO01 antibody (25 21B8 Diagast clone). Make up the volume to 4 ml with deionized water. Mix the solution end-to-end overnight to ensure coupling.

[0463] The coupled beads were then centrifuged at 2500 g for 5 min. Finally, the bead pellet was resuspended in storage buffer (phosphate buffered saline supplemented with 0.5% bovine serum albumin and 0.25 M sucrose) to restore the weight of the introduced bead solution.

[0464] 2. Preparation of the reaction device and testing procedures

[0465] The device is a molded plastic box in which a Porex HDPE hydrophobic membrane 0.6 mm thick and with a porosity between 9 and 12 μm, a drainage pad (cotton batt from Alan & co) and an absorbent pad (Cleanis) are assembled.

[0466] The porous membrane was hydrophilized with 1 μl of the surfactant solution listed above.

[0467] a.1.5% Triton X-100

[0468] b. Polyoxyethylene 10%

[0469] c. Nonyl-β-D-glucoside 50mM

[0470] Then 10 μl of beads coupled to the antibody at a concentration of 3% were spotted. The detection procedure was started by introducing 50 μl of red blood cells diluted at 1.5% in phosphate buffered saline supplemented with 0.00625% Tween-20 in each well. The reaction area was washed with 30 μl of phosphate buffered saline supplemented with 0.2% Tween-20.

[0471] 3. Results

[0472] Fig. 9The resulting image is shown, on which the presence of dark grey spots (corresponding to the red spots in the colour image) is observed, indicating the formation of immune complexes and therefore a positive reaction, whereas the presence of colourless spots reflects a negative reaction. Specificity is observed regardless of the surfactant used.

[0473] Example 7: Absorption and emission materials used in the device of the present invention

[0474] 1. Absorbent material

[0475] 1.1. Antibody conjugation

[0476] On 1 ml of homogenized 4% beads, add 200 μl of 20X phosphate buffered saline and dissolve 250 μl of 6D4 (anti-human IgM Diagast clone). Make up the volume to 4 ml with deionized water. Mix the solution end-over-end overnight to ensure coupling.

[0477] Excess reactive groups were capped by suspending 150 μl of 1 M ethanolamine in the bead antibody solution and mixed at room temperature for 2 hours. The AHG-coupled beads were then washed thoroughly twice with 3 ml of storage buffer (centrifugation at 2500 g for 5 min before and after washing). The bead pellet was resuspended with 2 ml of concentrated anti RH2 antibody (P3X255 13G8 Diagast clone) plus 2 ml of phosphate buffered saline. The solution was mixed end-to-end overnight to ensure coupling.

[0478] The coupled beads were then washed extensively twice with 3 ml of storage buffer (centrifugation at 2500 g for 5 min before and after washing). Finally, the bead pellet was resuspended in storage buffer to restore the weight of the introduced bead solution.

[0479] 1.2. Preparation of reaction apparatus and testing procedures

[0480] The apparatus is a molded plastic box in which a Porex HDPE hydrophobic membrane 0.6 mm thick and with a porosity between 9 and 12 μm, a drainage pad (NA7150 PES from Subrenat) and an absorbent pad (from Mc Arlaid reference below) are assembled.

[0481] The adsorbent materials used are as follows:

[0482] οT-499 or SCP-300TCF( Fig.10 a)

[0483] οT-099 or T-499( Fig.10 b)

[0484] οT-499 or T-183-5( Fig.10 c)

[0485] οT-499 or SCP-200-TCF( Fig.10 d)

[0486] The porous membrane was hydrophilized with 1 μl of surfactant solution (phosphate buffered saline supplemented with 0.5% Triton X-100; 1% green dye), then spotted with 10 μl of 1% antibody or negative control coupled beads, and the device was dried at 37°C, 20% humidity for 15 min before immediate use or long-term storage.

[0487] The assay was initiated by diluting red blood cells at 2.5% in chromasolcoombs (Diagast preparation) and introducing 50 μl followed by two 30 μl wash buffer (phosphate-buffered saline supplemented with 0.2% Tween 20) in each well.

[0488] Results

[0489] from Fig.10 a, b, c and d As can be seen, any of the absorbent layers tested ensures the detection of RH2-positive red blood cells (presence of red spots, visible as dark grey spots). Specificity is observed; colorless spots reflect RH2-negative red blood cells.

[0490] 2. Discharge materials

[0491] 2.1. Antibody conjugation

[0492] Dissolve 1 ml of concentrated anti-AB antibody (152D12 Diagast clone) on 1 ml of homogenized 4% beads. Make up the volume to 4 ml with phosphate buffered saline. Mix the solution end over end overnight to ensure coupling.

[0493] The coupled beads were then washed extensively twice with 3 ml of storage buffer (centrifugation at 2500 g for 5 min before and after washing). Finally, the bead pellet was resuspended in storage buffer to restore the weight of the introduced bead solution.

[0494] 2.2. Preparation of reaction device and testing procedures

[0495] The apparatus is a molded plastic box in which a Porex HDPE hydrophobic membrane 0.6 mm thick and with a porosity between 9 and 12 μm, a drain pad (referenced below) and an absorbent pad (Cleanis) are assembled.

[0496] The discharge materials are as follows:

[0497] o Cotton wool (from Alan & co), shown in Fig.11 a

[0498] οNT 9750HY (from Subrenat), shown in Fig.11 b,

[0499] οNT 9610HY (from Subrenat), shown in Fig.11 c

[0500] The porous membrane was hydrophilized with 1 μl of surfactant solution (phosphate buffered saline supplemented with 1% Triton X-100; 1% green dye), then spotted with 10 μl of 1% antibody or negative control coupled beads, and the device was dried at 37°C, 10% humidity for 15 min before immediate use or long-term storage.

[0501] The assay was initiated by diluting red blood cells at 2.5% in chromasolcoombs (Diagast preparation) and introducing 50 μl in each well followed by two 30 μl washes (phosphate buffered saline supplemented with 0.2% Tween 20).

[0502] Results

[0503] Fig.11 Any discharge pad is shown to ensure detection of B positive red blood cells (presence of red spots, visible as dark grey spots on the graph). Specificity is observed; colorless spots are obtained with O red blood cells.

[0504] Example 8: Blood typing and testing of a mixed population using the device of the present invention

[0505] 1. Antibody Conjugation

[0506] On 1 ml of homogenized 4% beads (Thermo Fisher latex aldehyde / sulfate 9 μm 4% w / v), add 100 μl of 20X phosphate buffered saline and dissolve 2 ml of anti-ABO01 antibody (91 13D10 Diagast clone) or anti-ABO02 antibody (96 21A8 Diagast clone). Make up the volume to 4 ml with deionized water. Mix the solution end-to-end overnight to ensure coupling.

[0507] Excess active groups were capped by suspending 150 μl of 1 M ethanolamine in the bead antibody solution and mixed at room temperature for 2 hours. The coupled beads were then washed thoroughly twice with 3 ml of storage buffer (centrifuged at 2500 g for 5 min before and after washing). Finally, the bead pellet was resuspended in storage buffer to restore the weight of the introduced bead solution.

[0508] 2. Preparation of the reaction device and testing procedures

[0509] The apparatus is a moulded plastic box in which a Porex HDPE hydrophobic membrane 0.6 mm thick and with a porosity between 9 and 12 μm, a drainage pad (cotton batt from Alan & co) and an absorbent pad (Cleanis) are assembled.

[0510] The porous membrane was hydrophilized with 1 μl of surfactant solution (phosphate buffered saline supplemented with 1% Triton X-100 and 1% green dye). Then 10 μl of 3% antibody-coupled beads were spotted. Before use, the device was dried at 37°C and 20% humidity for 15 min.

[0511] The detection procedure was initiated by introducing into each well 50 μl of red blood cells diluted at 0.15% in Chromasolcoombs supplemented with 0.00625% Tween20.

[0512] Mixed field A and B populations were generated by mixing A and O or B and O red blood cells pre-diluted at 0.15% in Chromasolcoombs at the desired concentrations (30% O with 70% A or B red blood cells or 50% O with 50% A or B red blood cells, respectively).

[0513] The first image was acquired before washing. The reaction area was then washed twice with 30 μl of phosphate buffered saline supplemented with 0.2% Tween-20. Images were acquired after each wash.

[0514] 3. Results

[0515] like Fig.12 As shown in a and c, before washing, all spots appear dark grey (or red). Specificity is achieved after the second wash: the presence of red spots (visible as dark grey spots) indicates the formation of immune complexes and is therefore a positive reaction, while the presence of colorless spots reflects a negative reaction.

[0516] Fig.12 b and d show that after intensity interpretation of the acquired images (subtraction of the signal before and after two washes), the distinction between native and mixed field populations was satisfied.

[0517] Example 9: Detection of weak RH1 antigen and extended phenotype

[0518] 1. Protein AGL coupling

[0519] On 1 ml of homogenized 4% beads, 50 μg of protein AGL was dissolved in 3 ml PBS 1X and added. The solution was mixed end-over-end overnight to ensure coupling.

[0520] The coupled beads were then centrifuged at 2500 g for 5 min. Finally, the bead pellet was resuspended in storage buffer (phosphate buffered saline supplemented with 0.5% bovine serum albumin and 0.25 M sucrose) to restore the weight of the introduced bead solution.

[0521] 2. Preparation of the reaction device and testing procedures

[0522] The device is a molded plastic box in which a Porex HDPE hydrophobic membrane 0.6 mm thick and with a porosity between 9 and 12 μm, a drainage pad (9610HY) and an absorbent pad (Cleanis) are assembled.

[0523] The porous membrane was hydrophilized with 1 μl of a 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) with AGL-coupled beads. Before use, the device was dried at 37°C, 20% humidity for 15 min.

[0524] The detection procedure was started by introducing 50 μl of the reaction (weak RH1 or extended phenotype) in each well; 50 μl of red blood cells diluted at 1.5% in PBS 1X supplemented with 0.00625% Tween20 were added.

[0525] The first image was acquired before washing. The reaction area was then washed with 30 μl of phosphate buffered saline supplemented with 0.2% Tween-20. The image was acquired after washing.

[0526] 3 Results

[0527] Fig.13 a shows the resulting image of weak RH1, on which the presence of dark grey spots (corresponding to red spots in the colour image) was observed, indicating the formation of immune complexes and therefore a positive reaction, whereas the presence of colourless spots reflects a negative reaction.

[0528] Fig.13 b shows the resulting image of the extended phenotype, on which the presence of dark grey spots (corresponding to red spots in the colour image) is observed, indicating the formation of immune complexes and therefore a positive reaction, whereas the presence of colourless spots reflects a negative reaction.

[0529] Example 10: Reverse blood typing by 6D4 antibody coupled to beads

[0530] 1. Reverse blood typing

[0531] 1.1. Antibody conjugation

[0532] On 1 ml of homogenized 4% beads (Thermo Fisher latex aldehyde / sulfate 9 μm 4% w / v), 3 ml of 1X phosphate buffered saline was added and 100 μg of CP6D4 antibody was dissolved. The solution was mixed end-over-end overnight to ensure coupling.

[0533] Excess active groups were capped by suspending 150 μl of 1 M ethanolamine in the bead antibody solution and mixed at room temperature for 2 hours. The coupled beads were then washed thoroughly twice with 3 ml of storage buffer (centrifuged at 2500 g for 5 min before and after washing). Finally, the bead pellet was resuspended in storage buffer to restore the weight of the introduced bead solution.

[0534] 1.2. Preparation of reaction apparatus and testing procedures

[0535] The device is a molded plastic box in which a Porex HDPE hydrophobic membrane 0.6 mm thick and with a porosity between 9 and 12 μm, a drainage pad (Subrenat 9610HY) and an absorbent pad (Cleanis) are assembled.

[0536] The porous membrane was hydrophilized with 0.5 μl of surfactant solution (phosphate buffered saline supplemented with 1.5% Triton X-100; 0.5% green dye). 5 μl of 1% antibody-coupled beads were spotted and the device was dried at 37°C, 10% humidity for 15 min before immediate use or long-term storage.

[0537] The test was initiated by incubating 30 μl of plasma and 20 μl of red blood cells (A1; B; A2; O) during 5 min. 25 μl followed by 30 μl of washing buffer (phosphate buffered saline supplemented with 0.2% Tween 20) were introduced in each well.

[0538] Results

[0539] Fig.14 The presence of dark grey spots (corresponding to red spots) is shown, indicating capture of IgM-sensitized red blood cells, thus a positive reverse typing reaction, whereas the presence of colourless spots reflects a negative reaction (unsensitized red blood cells).

[0540] Example 11: DAT (Direct Coombs Test) test using the device of the present invention

[0541] 1. Red blood cells pre-sensitized with antibodies

[0542] 1.1. Antibody conjugation

[0543] On 3 ml of homogenized 4% beads (Thermo Fisher latex aldehyde / sulfate 9 μm 4% w / v), 3 ml of anti-human globulin solution was dissolved. The AHGs studied were SA6532 (polyclonal rabbit antibody against human IgG), C5-1 (Diagast clone, mouse IgG against human IgG) and 188 33 (Diagast clone, mouse IgM against human IgG). The solution was mixed end-to-end for one hour to ensure coupling.

[0544] The bead suspension was centrifuged at 2500 g for 5 min. Excess active groups were capped by resuspending the bead pellet in 6 ml of 50 mM ethanolamine solution, followed by mixing for 30 min. The coupled beads were then washed thoroughly twice with 9 ml of storage buffer (centrifuged at 2500 g for 5 min before and after washing). Finally, the bead pellet was resuspended in storage buffer to restore the weight of the introduced bead solution.

[0545] 1.2. Preparation of reaction apparatus and testing procedures

[0546] The device is a molded plastic box in which a Porex HDPE hydrophobic membrane 0.6 mm thick and with a porosity between 9 and 12 μm, a drainage pad (Subrenat 9610HY) and an absorbent pad (Cleanis) are assembled.

[0547] The porous membrane was hydrophilized with 0.5 μl of surfactant solution (phosphate buffered saline supplemented with 1.5% Triton X-100; 0.5% green dye). 5 μl of 3% antibody-coupled beads were spotted and the device was dried at 37°C, 10% humidity for 15 min before immediate use or long-term storage.

[0548] Red blood cells were pre-sensitized in vitro with FY, KEL1, or RH1 IgG antibodies to simulate direct Coombs-positive red blood cells.

[0549] The test was initiated by diluting red blood cells (coombs negative or positive) at 0.25% in chromasolcoombs (Diagast preparation) supplemented with 0.00625% Tween 20. 50 μl followed by 30 μl of wash buffer (phosphate buffered saline supplemented with 0.2% Tween 20) were introduced in each well.

[0550] 2. Direct antiglobulin test with Anti-IgG and Anti-C3d

[0551] 2.1. Antibody conjugation

[0552] On 1 ml of homogenized 4% beads (Thermo Fisher latex aldehyde / sulfate 9 μm 4% w / v), 3 ml of 1X phosphate buffered saline was added and 100 μg of SA6532 or C7610 antibody was dissolved. The solution was mixed end-to-end overnight to ensure coupling. The coupled beads were then centrifuged at 2500 g for 5 min. Finally, the bead pellet was resuspended in storage buffer to recover the weight of the introduced bead solution.

[0553] 2.2. Preparation of reaction device and testing procedures

[0554] The device is a molded plastic box in which a Porex HDPE hydrophobic membrane 0.6 mm thick and with a porosity between 9 and 12 μm, a drainage pad (Subrenat 9610HY) and an absorbent pad (Cleanis) are assembled.

[0555] The porous membrane was hydrophilized with 0.5 μl of surfactant solution (phosphate buffered saline supplemented with 1.5% Triton X-100; 0.5% green dye). 5 μl of 4% antibody-coupled beads were spotted and the device was dried at 37°C, 10% humidity for 15 min before immediate use or long-term storage.

[0556] The test was initiated by diluting red blood cells (DAT negative or positive) at 1.5% in phosphate buffered saline supplemented with 0.00625% Tween 20. 50 μl followed by 30 μl of wash buffer (phosphate buffered saline supplemented with 0.2% Tween 20) were introduced in each well.

[0557] 3. DAT by antibody affinity protein

[0558] 3.1. Protein AGL conjugation

[0559] On 1 ml of homogenized 4% beads, 50 μg of protein AGL was dissolved in 3 ml PBS 1X and added. The solution was mixed end-over-end overnight to ensure coupling.

[0560] The coupled beads were then centrifuged at 2500 g for 5 min. Finally, the bead pellet was resuspended in storage buffer (phosphate buffered saline supplemented with 0.5% bovine serum albumin and 0.25 M sucrose) to restore the weight of the introduced bead solution.

[0561] 3.2. Preparation of reaction device and testing procedures

[0562] The device is a molded plastic box in which a Porex HDPE hydrophobic membrane 0.6 mm thick and with a porosity between 9 and 12 μm, a drainage pad (9610HY) and an absorbent pad (Cleanis) are assembled.

[0563] The porous membrane was hydrophilized with 1 μl of a 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) with AGL-coupled beads. Before use, the device was dried at 37°C, 20% humidity for 15 min.

[0564] The detection procedure was started by incubating 20 μl of the reaction (DAT) in the wells; adding 50 μl of red blood cells diluted at 3% in PBS 1X supplemented with 0.00625% Tween 20 to the wells; adding 20 μl of IAT solution 2 buffer. 120 μl of the mixture was transferred to the wells of the cartridge.

[0565] Acquire the first image before washing. Then wash the reaction area with 40 μl of IAT solution 2 buffer. Acquire the image after washing.

[0566] 4. Results

[0567] Fig.15 a shows the presence of dark grey spots (corresponding to red spots), indicating capture of IgG sensitized red blood cells, thus a positive direct Coombs reaction, whereas the presence of colorless spots reflects a negative reaction (unsensitized red blood cells).

[0568] Fig.15 b shows the presence of dark grey spots (corresponding to red spots), indicating capture of IgG or / and C3D-sensitized red blood cells and thus a positive reaction, whereas the presence of colourless spots reflects a negative reaction (unsensitized red blood cells).

[0569] Fig.15 c shows the resulting image on which the presence of dark grey spots (corresponding to red spots in the colour image) is observed, indicating the formation of immune complexes and therefore a positive reaction, whereas the presence of colourless spots reflects a negative reaction.

[0570] Example 12: Detection of antibodies specific to malarial antigens using the device of the present invention

[0571] 1. Antigen coupling

[0572] On 1 ml of homogenized 4% beads, 100 μg of Antigen Mal003 was dissolved in 3 ml PBS 1X and added. The solution was mixed end-over-end overnight to ensure coupling.

[0573] The coupled beads were then centrifuged at 2500 g for 5 min. Finally, the bead pellet was resuspended in storage buffer (phosphate buffered saline supplemented with 0.5% bovine serum albumin and 0.25 M sucrose) to restore the weight of the introduced bead solution.

[0574] 2. Antibody conjugation for detection

[0575] On 1 ml of homogenized 4% beads 1 μm, 200 μg of Antibodies 6D4 were dissolved in 3 ml PBS 1X and added. The solution was mixed end-over-end overnight to ensure coupling.

[0576] The coupled beads were then centrifuged at 5000 g for 5 min. Finally, the bead pellet was resuspended in storage buffer (phosphate buffered saline supplemented with 0.5% bovine serum albumin and 0.25 M sucrose) to restore the weight of the introduced bead solution.

[0577] 3. Preparation of the Reaction Apparatus and Detection Procedure Using HRP

[0578] The device is a molded plastic box in which a Porex HDPE hydrophobic membrane 0.6 mm thick and with a porosity between 9 and 12 μm, a drainage pad (9610HY) and an absorbent pad (Cleanis) are assembled.

[0579] The porous membrane was hydrophilized with 0.5 μl of surfactant solution (phosphate buffered saline supplemented with 2% Triton X-100 and 1% green dye). Then 2 μl of 4% beads coupled to the antigen Mal003 were spotted. Before use, the device was dried at 37°C and 20% humidity for 15 min.

[0580] Start the detection procedure by introducing 100 μl of sample (kit ab178649) in each well. Add 100 μl of antibody Anti-IgG and / or Anti-IgM HRP conjugate (kit ab178649). Acquire the first image before washing. Then wash the reaction area with 30 μl of phosphate buffered saline supplemented with 0.2% Tween-20. Then wash the reaction area with 30 μl of display buffer (TMB substrate (kit ab178649)). Acquire the image after 15 min.

[0581] 4. Preparation of the Reaction Apparatus and Detection Procedure Using Beads

[0582] The device is a molded plastic box in which a Porex HDPE hydrophobic membrane 0.6 mm thick and with a porosity between 9 and 12 μm, a drainage pad (9610HY) and an absorbent pad (Cleanis) are assembled.

[0583] The porous membrane was hydrophilized with 0.5 μl of surfactant solution (phosphate buffered saline supplemented with 2% Triton X-100 and 1% green dye). Then 2 μl of 4% beads coupled to the antigen Mal003 were spotted. Before use, the device was dried at 37°C and 20% humidity for 15 min.

[0584] The detection procedure was started by introducing 100 μl of sample (kit ab178649) in each well. 30 μl of 0.1% antibody 6D4 beads 1 μm were added. The first image before washing was acquired. The reaction area was then washed with 120 μl of phosphate buffered saline supplemented with 0.2% Tween-20. Images were acquired after washing.

[0585] 5. Preparation of the Reaction Apparatus and Detection Procedure Using Colloidal Gold Nanoparticles

[0586] The device is a molded plastic box in which a Porex HDPE hydrophobic membrane 0.6 mm thick and with a porosity between 9 and 12 μm, a drainage pad (9610HY) and an absorbent pad (Cleanis) are assembled.

[0587] The porous membrane was hydrophilized with 0.5 μl of surfactant solution (phosphate buffered saline supplemented with 2% Triton X-100 and 1% green dye). Then 5 μl of 4% beads coupled to the antigen Mal003 were spotted. Before use, the device was dried at 37°C and 20% humidity for 15 min.

[0588] The detection procedure was started by introducing 50 μl of sample (kit ab178649) in each well. 10 μl of anti-human IgA, IgG, IgM 60 nm gold conjugate (AC-60-14-10) was added. The first image before washing was acquired. The reaction area was then washed with 50 μl of phosphate buffered saline supplemented with 0.2% Tween-20. Images were acquired after washing.

[0589] 6. Results

[0590] Fig.16 a shows the resulting image on which the presence of dark grey spots is observed, indicating the formation of immune complexes and thus a positive reaction, whereas the presence of colourless spots reflects a negative reaction and a pattern with grey intensity.

[0591] Fig.16 b shows the resulting image on which the presence of dark grey spots is observed, indicating the formation of immune complexes and therefore a positive reaction, whereas the presence of colourless spots reflects a negative reaction and a pattern with grey intensity.

[0592] Fig.16c shows the resulting image on which the presence of dark grey spots is observed, indicating the formation of immune complexes and therefore a positive reaction, whereas the presence of colourless spots reflects a negative reaction and a pattern with grey intensity.

[0593] Example 13: Using the device of the present invention with 1 μm-red beads as a detection method to detect antibodies against Treponema pallidum Antibodies with specificity

[0594] 1. Antigen coupling

[0595] On 1 ml of homogenized 4% beads, 100 μg of p15, p17, p47 Treponema pallidum antigens (30-AT76) were dissolved in 3 ml PBS 1X and added. The solution was mixed end-over-end overnight to ensure coupling.

[0596] The coupled beads were then centrifuged at 2500 g for 5 min. Finally, the bead pellet was resuspended in storage buffer (phosphate buffered saline supplemented with 0.5% bovine serum albumin and 0.25 M sucrose) to restore the weight of the introduced bead solution.

[0597] 2. Antibody conjugation for detection

[0598] On 1 ml of homogenized 4% beads 1 μm, 200 μg of AGL protein was dissolved in 3 ml PBS 1X and added. The solution was mixed end-over-end overnight to ensure coupling.

[0599] The coupled beads were then centrifuged at 5000 g for 5 min. Finally, the bead pellet was resuspended in storage buffer (phosphate buffered saline supplemented with 0.5% bovine serum albumin and 0.25 M sucrose) to restore the weight of the introduced bead solution.

[0600] 3. Preparation of reaction device and testing procedures

[0601] The device is a molded plastic box in which a Porex HDPE hydrophobic membrane 0.6 mm thick and with a porosity between 9 and 12 μm, a drainage pad (9610HY) and an absorbent pad (Cleanis) are assembled.

[0602] The porous membrane was hydrophilized with 0.5 μl of surfactant solution (phosphate buffered saline supplemented with 2% Triton X-100 and 1% green dye). Then 2 μl of 4% beads coupled with p15, p17, and p47 Treponema pallidum antigens were applied. Before use, the device was dried at 37°C and 20% humidity for 15 min.

[0603] The detection procedure was started by introducing 10 μl of sample (Treponema pallidum antibody 20-TR89) in each well. 30 μl of 0.1% AGL protein beads 1 μm were added. The first image before washing was acquired. The reaction area was then washed with 120 μl of phosphate buffered saline supplemented with 0.2% Tween-20. Images were acquired after washing.

[0604] 4. Results

[0605] Fig.17 The resulting image is shown, on which the presence of dark grey spots is observed, indicating the formation of immune complexes and therefore a positive reaction, whereas the presence of colourless spots reflects a negative reaction and a pattern with grey intensity.

[0606] Example 14: Detection of hepatitis B virus antigen using the device of the present invention using the 1 μm red bead detection method (HbsAg) has specific antibodies

[0607] 1. Antigen coupling

[0608] On 1 ml of homogenized 4% beads, 100 μg of HBs Antigen (30-AH15) was dissolved in 3 ml PBS 1X and added. The solution was mixed end-over-end overnight to ensure coupling.

[0609] The coupled beads were then centrifuged at 2500 g for 5 min. Finally, the bead pellet was resuspended in storage buffer (phosphate buffered saline supplemented with 0.5% bovine serum albumin and 0.25 M sucrose) to restore the weight of the introduced bead solution.

[0610] 2. Antibody conjugation for detection

[0611] On 1 ml of homogenized 4% beads 1 μm, 200 μg of AGL protein was dissolved in 3 ml PBS 1X and added. The solution was mixed end-over-end overnight to ensure coupling.

[0612] The coupled beads were then centrifuged at 5000 g for 5 min. Finally, the bead pellet was resuspended in storage buffer (phosphate buffered saline supplemented with 0.5% bovine serum albumin and 0.25 M sucrose) to restore the weight of the introduced bead solution.

[0613] 3. Preparation of reaction device and testing procedures

[0614] The device is a molded plastic box in which a Porex HDPE hydrophobic membrane 0.6 mm thick and with a porosity between 9 and 12 μm, a drainage pad (9610HY) and an absorbent pad (Cleanis) are assembled.

[0615] The porous membrane was hydrophilized with 0.5 μl of surfactant solution (phosphate buffered saline supplemented with 2% Triton X-100 and 1% green dye). Then 2 μl of 4% beads coupled to HBs antigen were applied. Before use, the device was dried at 37°C and 20% humidity for 15 min.

[0616] The detection procedure was started by introducing 10 μl of sample (HBsAg antibody 10-H05G) in each well. 30 μl of 0.1% AGL protein beads 1 μm were added. The first image before washing was acquired. The reaction area was then washed with 120 μl of phosphate buffered saline supplemented with 0.2% Tween-20. Images were acquired after washing.

[0617] 4. Results

[0618] Fig.18 The resulting image is shown, on which the presence of dark grey spots is observed, indicating the formation of immune complexes and therefore a positive reaction, whereas the presence of colourless spots reflects a negative reaction and a pattern with grey intensity.

[0619] Example 15: Detection of Hepatitis B Virus Antigen Using the Device of the Present Invention

[0620] 1. Antigen coupling

[0621] On 1 ml of homogenized 4% beads, 100 μg of HBs Antibody (10-H05G) was dissolved in 3 ml of PBS 1X and added. The solution was mixed end-over-end overnight to ensure coupling.

[0622] The coupled beads were then centrifuged at 2500 g for 5 min. Finally, the bead pellet was resuspended in storage buffer (phosphate buffered saline supplemented with 0.5% bovine serum albumin and 0.25 M sucrose) to restore the weight of the introduced bead solution.

[0623] 2. Antibody conjugation for detection

[0624] On 1 ml of homogenized 4% beads 1 μm, 200 μg of HBs Antibody (10-H05H) was dissolved in 3 ml PBS 1X and added. The solution was mixed end-over-end overnight to ensure coupling.

[0625] The coupled beads were then centrifuged at 5000 g for 5 min. Finally, the bead pellet was resuspended in storage buffer (phosphate buffered saline supplemented with 0.5% bovine serum albumin and 0.25 M sucrose) to restore the weight of the introduced bead solution.

[0626] 3. Preparation of the reaction apparatus and detection procedure using the HRP method as the detection method

[0627] The device is a molded plastic box in which a Porex HDPE hydrophobic membrane 0.6 mm thick and with a porosity between 9 and 12 μm, a drainage pad (9610HY) and an absorbent pad (Cleanis) are assembled.

[0628] The porous membrane was hydrophilized with 0.5 μl of surfactant solution (phosphate buffered saline supplemented with 2% Triton X-100 and 1% green dye). Then 2 μl of 4% beads coupled with antigen HBs Antibody (10-H05G) were applied. Before use, the device was dried at 37°C and 20% humidity for 15 min.

[0629] Start the detection procedure by introducing 50 μl of sample (Kit 1701-12) in each well. Add 50 μl of Anti-HBs Antigen HRP Conjugate (Kit 1701-12). Acquire the first image before washing. Then wash the reaction area with 100 μl of display buffer (TMB substrate (Kit 1701-12)). Acquire the image after 15 min.

[0630] 4. Preparation of the Reaction Apparatus and Detection Procedure Using the Bead Method

[0631] The device is a molded plastic box in which a Porex HDPE hydrophobic membrane 0.6 mm thick and with a porosity between 9 and 12 μm, a drainage pad (9610HY) and an absorbent pad (Cleanis) are assembled.

[0632] The porous membrane was hydrophilized with 0.5 μl of surfactant solution (phosphate buffered saline supplemented with 2% Triton X-100 and 1% green dye). Then 2 μl of 4% beads coupled to HBs Antibody (10-H05G) were applied. Before use, the device was dried at 37°C and 20% humidity for 15 min.

[0633] Start the detection procedure by introducing 10 μl of sample HBs antigen (kit 1701-12) in each well. Add 100 μl of 0.025% HBs Antibody (10-H05H) beads 1 μm. Acquire the first image before washing. Then wash the reaction area with 120 μl of phosphate buffered saline supplemented with 0.2% Tween-20. Acquire the image after washing.

[0634] 5. Results

[0635] Fig.19 a shows the resulting image on which the presence of dark grey spots is observed, indicating the formation of immune complexes and thus a positive reaction, whereas the presence of colourless spots reflects a negative reaction and a pattern with grey intensity.

[0636] Fig.19 b shows the resulting image on which the presence of dark grey spots is observed, indicating the formation of immune complexes and therefore a positive reaction, whereas the presence of colourless spots reflects a negative reaction and a pattern with grey intensity.

Claims

1. A vertical flow-based in vitro diagnostic device for detecting and / or identifying antigens and / or antibodies from a biological fluid sample, comprising: - a support, and - a hydrophobic porous membrane arranged in the support, comprising at least one hydrophilic reaction area intended to receive the sample, the surface of the hydrophilic reaction area being smaller than the surface of the hydrophobic porous membrane, the surface of the hydrophilic reaction area comprising at least one antibody or antigen, wherein the antibody or antigen is immobilized or adsorbed on the surface of beads, wherein the size of the beads is larger than the size of the pores of the hydrophobic membrane.

2. The in vitro diagnostic device of claim 1, wherein the biological fluid sample is a blood sample or a blood component sample.

3. The in vitro diagnostic device of claim 1, wherein the antibody is selected from the group consisting of anti-red blood cell antibodies, anti-platelet antibodies, antibodies to viral antigens, antibodies to bacterial antigens, and antibodies to parasite antigens.

4. The in vitro diagnostic device of claim 1, wherein the antigen is selected from the group consisting of a red blood cell antigen, a platelet antigen, a viral antigen, a bacterial antigen, and a parasite antigen.

5. The in vitro diagnostic device of claim 3 or 4, wherein the antibody to the viral antigen and the viral antigen are from hepatitis B virus or hepatitis C virus.

6. The in vitro diagnostic device of claim 3 or 4, wherein the antibody to the bacterial antigen and the bacterial antigen are from the genus Treponema Treponema .

7. The in vitro diagnostic device of claim 3 or 4, wherein the antibody to the parasite antigen and the parasite antigen are from the genus Plasmodium Plasmodium .

8. The in vitro diagnostic device according to any one of claims 1 to 4, wherein the antibody or antigen is directly or indirectly immobilized or adsorbed on the bead surface.

9. The in vitro diagnostic device according to any one of claims 1 to 4, wherein the antibody or antigen is immobilized on the bead surface indirectly via another antibody.

10. The in vitro diagnostic device of claim 9, wherein the other antibody is immunoglobulin IgM or IgG.

11. The in vitro diagnostic device according to any one of claims 1 to 4, wherein the antibody is immobilized on the bead surface indirectly via a ligand selected from proteins having affinity for the antibody.

12. The in vitro diagnostic device of claim 11, wherein the ligand is protein A, protein G or protein L.

13. The in vitro diagnostic device according to any one of claims 1 to 4, wherein the bead surface comprises at least one chemical group selected from an aldehyde group, a chloromethyl group, an NHS group and a carboxyl group.

14. The in vitro diagnostic device according to any one of claims 1 to 4, wherein the average size of the beads is between 1 µm and 130 µm.

15. The in vitro diagnostic device of claim 14, wherein the average size of the beads is between 5 µm and 50 µm.

16. The in vitro diagnostic device of claim 15, wherein the average size of the beads is 9 µm.

17. An in vitro diagnostic device according to any one of claims 1 to 4, wherein the beads are used in a concentration of 1% to 10% of the solution in which they are suspended, and wherein the deposited volume of the solution is between 1 and 100 μl.

18. The in vitro diagnostic device according to any one of claims 1 to 4, wherein the thickness of the hydrophobic porous membrane is between 0.4 mm and 2 mm.

19. The in vitro diagnostic device according to any one of claims 1 to 4, wherein the hydrophilic reaction region of the hydrophobic porous membrane is rendered hydrophilic by a surfactant.

20. The in vitro diagnostic device of claim 19, wherein the surfactant is used at a concentration of 0.01 to 5% w / v of the solution, and wherein 0.1 to 5 μl of the solution containing the surfactant is placed on the hydrophobic porous membrane.

21. The in vitro diagnostic device according to any one of claims 1 to 4, further comprising at least one layer arranged below the porous membrane, which is an absorbent layer.

22. The in vitro diagnostic device according to any one of claims 1 to 4, further comprising at least one further layer arranged between the hydrophobic porous membrane and the absorbent layer, the at least one further layer being made of a drainage material.

23. Use of the in vitro device according to any one of claims 1 to 22 for determining and / or identifying: - at least one antigen selected from the group consisting of red blood cell antigens, 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-parasite antibodies; It is derived from a biological fluid sample.

24. An in vitro method for detecting and / or identifying red blood cell antigens and / or in vivo sensitized red blood cell and / or platelet antigens from a blood sample or a blood component sample, comprising the following steps: - adding a solution containing the sample onto a reaction area of ​​the in vitro device according to any one of claims 1 to 3 and 8 to 22, - Reading before washing as sample loading control, - depositing a cleaning solution onto the reaction area, and - If a red or pink spot appears in the reaction area, the presence of the antigen or the blood cell is determined by determining the presence of an antigen / antibody interaction, or if a colorless spot appears in the reaction area, the absence of the antigen or the blood cell is determined by determining the absence of an antigen / antibody reaction.

25. An in vitro method for detecting and / or identifying anti-red blood cell antibodies from a blood sample or a blood component sample, comprising the following steps: - incubate the sample to be tested with a buffer and a red blood cell test substance of known phenotype, - depositing the mixture on a reaction zone of an in vitro device according to any one of claims 1, 4 and 8 to 22, - depositing a cleaning solution onto the reaction area, and - The presence of anti-red blood cell antibodies is determined by determining the presence of an antibody / antigen reaction if a red or pink spot appears in the reaction area, or the absence of said antibodies is determined by determining the absence of an antibody / antigen reaction if a colorless spot appears in the reaction area.

26. An in vitro method for detecting and / or identifying anti-platelet antibodies from a blood sample or a blood component sample, comprising the steps of: - incubation of the sample to be tested with a buffer and a platelet test substance of known antigenicity, or incubation of the recipient's plasma or serum with the donor's platelets, - depositing the mixture on a reaction zone of an in vitro device according to any one of claims 1, 4 and 8 to 22, - depositing a cleaning solution onto the reaction area, and - The presence of antiplatelet antibodies is determined by determining the presence of antibody / antigen interaction if a red or pink spot appears in the reaction area, or the absence of said antibodies is determined by determining the absence of antigen / antibody interaction if a colorless spot appears in the reaction area.

27. An in vitro method for detecting and / or identifying an antigen selected from the group consisting of a viral antigen, a bacterial antigen and a parasite antigen from a biological fluid sample, comprising the steps of: - adding a solution containing the sample onto a reaction area of ​​an in vitro device according to any one of claims 1 to 3 and 5 to 22, and - If a colored spot appears in the reaction area, the presence of the antigen is determined by determining the presence of an antigen / antibody interaction, or if a colorless spot appears in the reaction area, the absence of the antigen is determined by determining the absence of an antigen / antibody reaction.

28. The in vitro method according to claim 27, further comprising the steps of: - Incubate the sample to be tested with the buffer, - after adding a mixture of sample and buffer onto the reaction area of ​​the in vitro device according to any one of claims 1 to 3 and 5 to 22, reading before washing as a sample loading control, and - Deposit cleaning solution onto the reaction area.

29. The in vitro method according to claim 27, wherein the biological fluid sample is a blood sample or a blood component sample.

30. An in vitro method for detecting and / or identifying an antibody selected from the group consisting of an anti-viral antibody, an anti-bacterial antibody and an anti-parasitic antibody from a biological sample, comprising the following steps: - adding a solution containing the sample onto a reaction area of ​​the in vitro device according to any one of claims 1, 4 to 22, and - If a colored spot appears in the reaction area, the presence of the antibody is determined by determining the presence of an antigen / antibody interaction, or if a colorless spot appears in the reaction area, the absence of the antibody is determined by determining the absence of an antigen / antibody reaction.

31. The in vitro method according to claim 30, further comprising the steps of: - Incubate the sample to be tested with the buffer, - after adding a mixture of sample and buffer to the reaction area of ​​the in vitro device according to any one of claims 1, 4 to 22, reading before washing as a sample loading control, and - Deposit cleaning solution onto the reaction area.

32. The in vitro method according to claim 30, wherein the biological sample is a blood sample or a blood component sample.

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