TW201209413A - Immunomagnetic complex and its use in red blood cell grouping/phenotyping - Google Patents

Abstract

The present invention relates to a reagent for RBC grouping/phenotyping comprising a suspension of magnetic particles or beads coated with an antiglobulin saturated with RBC anti-antigen antibodies, and a kit or device containing the above. The present invention also relates to an RBC grouping/phenotyping method using such a suspension of magnetic particles or beads according to the present invention.

Description

201209413 VI. Description of the Invention: [Technical Field] The present invention relates to a reagent for blood type determination/phenotype verification of red blood cells (RBC), and a kit or device containing the same, wherein the reagent comprises: A globular coated magnetic particle or suspension of magnetic beads which is saturated with an anti-erythrocyte antigen antibody. The present invention also relates to a method for performing blood type assay/phenotypic assay of red blood cells using such a magnetic particle or magnetic bead suspension of the present invention. [Prior Art] In the clinical blood transfusion practice, erythrocyte phenotypic examination is performed on blood donors and blood donors, that is, screening and identification of blood group antigens on the surface of red blood cells (excluding the ΑΒ0 system, the ΑΒ0 system also searches for the corresponding conventional antibodies). For the parent jk and the donor ik, there are three levels of red blood cell phenotype identification to provide red blood cell concentrates that are consistent with the risk profile of the recipient: -ΑΒ0 and rhesus monkey (Rhesus, hereinafter referred to as Rh) standard blood type Determination (presence or absence of D antigen); - determination of Rh and Kell phenotype (presence of C, E, C, 6 and κ antigens); and - determination of extended (or expanded) phenotype , that is, the presence or absence of antigens from the Duffy system (Fya, Fyb), the Kidd system (Jka, Jkb) and the MNSs system (S and s); - depending on the type of risk and / or Other antibodies can be investigated by detecting unregular antibodies in the serum of the recipient. The techniques used for phenotypic assays are usually made up of the use of a suitable antibody, 95288 3 201209413, using test serum to explore the presence or absence of the relevant antigen. The antibodies contained in the serum for such tests should preferably be a lectin (丨(4) or IgA) such that when the red blood cells to be assayed have an antigen corresponding to the antibody of the test blood, the red The ball will be agglomerated in whole or in part. However, an antibody for non-lectin test (I gG type) can also be used. In this case, agglutination is produced by anti-human immunoglobulin, which is more visible in the case of centrifugation and re-floating of packed red bl〇〇d cells ("indirect library" M (c〇〇mbs) technology"). This test is referred to as the "indirect Coom" system because the first stage consists of 37 red blood cells and test IgG. (: cultivating together. After incubation, the cells are washed by centrifugation = removal of excess unfixed antibody and maximization of anti-human globulin (ahg). AHG (anti- The human globulin is immobilized on the test carcass such that the red blood cells are agglomerated by forming a macromolecular complex which can be promoted by centrifugation and resuspension of the red blood cells which are tightly pressed. Or in various variations of the technique of non-conventional antibody screening (IAS) '(10) a general method of detecting an analyte that can be knotted in a sample by using magnetic particles, to the analyte of the cell, notably, Equivalent methods do not require centrifugation of 'difficult-based techniques from IAS' such as anti-ball technology (ie, by agglutination between the Kulm test or by immunoadsorption on a solid phase). Type assay or similarly for IAS, the sensitized red blood cells must be flushed when needed to remove the non-specific antibodies that recognize the anti-immunoglobulin used in the next step. 8

S 4 201209413 is mainly based on the cost, size and operation of the centrifuge. When the project fully automates the process, it is always difficult to carry out the centrifugation step. Magnetic particles have been used for the detection of ligand-receptor or antibody-antigen complexes for many years. Examples thereof include the methods described in the following patent documents: - Document W092/17781, which describes a method of determining the presence of a ligand in a sample, wherein the incubation is carried out with a substance (such as an antibody) that binds to the ligand. Magnetic latex particles, which may have different colors. The magnetic field is then applied to the medium to finally observe the presence or absence of agglutination; or - document EP 0 426 170 'which describes a method for determining the presence of a ligand in a sample, the method comprising: cultivating an already used antigen or Body-bound antibody-sensitized magnetic gelatin particles. The magnetic field is then applied to the incubation medium to finally observe the presence or absence of gelation; the method is characterized in that the sliding state of the magnetic particles is observed after tilting the container, which has a v-shaped bottom in a microplate (micr〇plate) The cup-shaped recess is particularly noticeable. These magnetic particles have been used in immunohematology such as phenotypic assays and/or IAS. Documents relating to such applications are, for example: - document W02005121805 'which describes a method for blood type determination/phenotypic assay and non-conventional lectin screening, in which a liquid aqueous solution of ferrofluid is used; - document EP0351857, And an immunoassay method in which a magnetization label such as an antibody or an anti-protoxin immobilized on a latex magnetic bead is used; _document EP0528708' describes a detection method which may be present in a sample by using a latex magnetic bead for immunoadsorption In the case of biological substances, it is tested in 95288 5 201209413; and 2 documents EPG23G768, which refers to the substances contained in the sample and the substance contained in the sample. ==::\: Methodology - Although using magnetic beads or modified The technology of liquid magnetic material eliminates the need for centrifugation 'but it needs to be (4) coated with blood for inspection or phenotypic determination. Solid phase The coated solid phase lacks stability or must be prepared by the end user. Furthermore, see document EP 768. When using a magnetic field to make certain magnetic compounds non-specifically coaggregate gamma cells, it will never be possible to read "the red color caused by the presence of antibodies against red A-filaments." Ball-specific agglutination" (a reference technique in the field of blood transfusion). The desired one is a quick and easy way to perform on a practical and accessible support such as a microplate, i.e., the centrifugation step is replaced by the application of a magnetic field. The method can thus be fully automated, and the blood type assay/phenotype assay of red blood cells can be guided by reading antibodies of the IgG type assay (IgG itself is non-aggregating, but it is readily available and provides a wide range of specificity). Specific agglutination of the sputum is performed, and the final image (result) is not significantly disturbed. This method can also be applied to the Simonin test (indirect test) for the ΑΒ0 phenotype. This method would be more advantageous if the magnetic compound used does not cause non-specific agglutination of red blood cells and the phenotypic assay can be exempted from the need for agglutinated serum (less expensive, more expensive). Object of the Invention [Summary of the Invention]

95288 S 201209413 The present inventors have shown that red blood cells are contacted with a dilute aqueous suspension of magnetic particles (magnetic particles or beads) coated with an antiglobulin such as anti-human globulin (AHG), wherein the AHG has been previously countered The antibody of the red blood cell type/phenotype antigen is saturated; so that regardless of the anti-erythrocyte type/phenotype antigen resistance, the agglutination nature (ie, not f is IgG type or IgM type), specificity occurs after application of a magnetic field Agglutination. Furthermore, the reagent can be subjected to blood type testing or phenotypic testing without centrifugation, resulting in complete automation of the procedure. Finally, the inventors have shown that such ready-to-use reagents are stable and can be stored in use J. The present inventors have also unexpectedly shown that the magnetic beads or the presence of the beads in the dilute suspension; j; will significantly read the specific agglutination. In fact, this agglutination can be easily seen by an automated reading system that can detect the presence of red blood collections. Finally, in the case of phenotypic assays, such reagents allow the use of type (4) against erythrocyte type A/phenotypic antibody, which makes it possible to purchase by specific agglutination. Blood grouping is used in the AB blood group system; the term "phenotype test" refers to other blood group systems. The invention achieves: the red blood cell blood type test phenotype test based on the test (4), the test does not need to be carried out after 3: the rinsing step after the cultivation to clear the specific IgG of the f1, or the centrifugation step to stimulate the red gold ball The agglomeration of the complex formed by the name. [Embodiment] The present invention relates to a blood type test for the use of (4) agents of Lisi Cai; 95288 7 201209413 and a method for phenotyping. The present invention uses magnetic particles coated with anti-human globulin which is previously saturated with 1 gG which is specific for an antigen considered to be in red blood cells (refer to Fig. 1). This makes it possible to perform an indirect Cumm test without rinsing or centrifugation, since the specific IgG has been immobilized on the antiglobulin without rinsing, and in addition, since the red blood cells are fixed to the immunomagnetic complex The application of a magnetic field will pull the red blood cells toward the bottom of the hole to increase their agglutination. Stirring after the magnetization phase allows the red blood cells that are tightly pressed together to resuspend and display agglomerates. This method allows blood group examination and phenotypic assays for all antigens present on the surface of red blood cells as long as their antibodies are available. This type of test can also be performed using IgM to enhance its cohesion. The anti-IgM type anti-human globulin must be immobilized on the magnetic beads and saturated with IgM specific for the antigen to be detected. Two types of beads may also be mixed in the same ready-to-use reagent, wherein each type of beads is coupled to an antiglobulin and a specific antibody; by this combination, the detection of a variant or a weak antigen can be improved. This is especially true when studying weak D antigens, where a mixture of multiple strains is often used to maximize the identification of all weak D antigens and variant D antigens. Thus, it would be easy to perform specific agglutination and comprehensively characterize the individual's red blood cell phenotype, and allow the use of any type of antibody regardless of its isotype. Thus, the macromolecule, i.e., the immunomagnetic complex, is subjected to a magnetic field after application of a magnetic field to agglutinate the red blood globules which express the desired antigen. Generally used centrifugal steps

8 95288 S 201209413 This is followed by a magnetization step instead of increasing agglutination. Therefore, the object of the present invention is a suspension of magnetic particles or magnetic beads, characterized in that the magnetic particles are coated on the surface with an anti-globulin complex, which is an anti-human globulin ( An AHG }/antibody is preferred, and the antibody in the complex specifically targets an erythrocyte blood group/phenotype antigen (this antibody is referred to as an antibody against a red blood cell blood type/phenotype antigen). AG) is an anti-species globulin (ie, 杬-species AG) of a given species, and the antibody against the erythrocyte blood group/phenotype antigen will be the source of the species to which the AG is calibrated. For example, and preferably, when AG In the case of anti-human globulin (AHG), the anti-system in the complex is an antibody against human hematopoietic/phenotypic antigen from humans. However, 'AG can also be a mouse type AG, in which case the antibody It is an antibody derived from a mouse. This concept also applies to the case where AG is a rat type or any other mammalian type AG. In a preferred embodiment of the invention, AGH may be a protein A or a protein G, or any energy Specific binding of antibodies to their Fc fragments White matter or polypeptide substitution 'where the Fc fragment is preferably a bifunctional polypeptide (two antibodies can be bound by the bifunctional polypeptide h in the present invention 'the term "magnetic particle" or "magnetic bead" refers to the same guest' That is, a spherical magnetic bead having a diameter of at least 0.5 micrometers (//m) and a maximum of 10 micrometers (unlike a liquid magnetic substance, the liquid magnetic substance is not within the range defined by the magnetic beads). The supply of the magnetic beads of the present invention can be provided. For example, Ademtech (33600 Pessac 'France), which is sold for about 1〇〇 to 500 9 95288 201209413

A company of nanometer (nm) diameter magnetic beads that can be functionalized with acid or amine functionality. The company also sells the protocols and reagents needed to graft these functionalities. These magnetic beads are composed of more than 50% of a core made of a ferromagnetic compound (iron oxide type) and then coated with polystyrene. Another supplier is Bioclone Inc. (San Diego, Calif.), which has all types of suitably functionalized magnetic beads. The third potential supplier is Dynal (Invitrogen, USA), which offers a wide range of DynabeadsTM, especially microbeads activated by streptavidin, toluene or ruthenium. Merck-Chimie SAS (Fontenay Sous Bois, France) has various sizes of micromagnetic particles (ESTAP0Rtm) ranging from 200 nm to 1.5 μm, which are basically made of polystyrene or diethylbenzene and include more than 50 % of ferrites e such particles may or may not be functionalized, for example, may be functionalized with a carboxyl group, an amine group or a tosyl group. These particles are prepared by a method involving the polymerization of styrene in the presence of a ferromagnetic compound. Another company is jSR Micr(R) (Japan), which markets magnetic particles of various sizes from 1 micron to 3 microns, which are activated by streptavidin or have carboxyl or toluenesulfonyl functions. The magnetic beads of JSR are composed of core particles coated with a magnetic substance, and the magnetic beads themselves are coated with a monomer to encapsulate the magnetic substance. Among these available beads, 1 micron hydrophobic beads are preferred. It has an iron content of about 15 nanomolar (_丨) line-based functionality per milligram of beads. This (four) particle is characterized by its ability to be coupled by physical adsorption or chemical coupling. The magnetic beads suitable for use in the present invention are superparamagnetic and have a magnetic particle size between nanometer and 1.5«. These magnetic beads are preferably hydrophobic and are sulfhydryl or

10 95288 S 201209413 Toluenesulfonyl functionalization. The average iron content in the magnetic beads is about 45% for the magnetic beads and about 3 〇 0/〇 for the sulfonated magnetic beads. Preferably, the suspension of magnetic particles according to the present invention is characterized in that the AHG is selected from the group consisting of anti-IgG type or anti-IgM type AHG. Further preferably, the suspension of the magnetic particles according to the present invention is characterized in that when the AHG is of the anti-IgG type, the antibody against the erythrocyte blood type/phenotype antigen is of the IgG type, and when the AHG is of the anti-1 gM type. In this case, the antibody against the erythrocyte gold/phenotype antigen is of the I gM type. Again preferably, the suspension of magnetic particles according to the present invention is characterized by saturation of the antibody against the erythrocyte/phenotype antigen. In the present invention, the term "antibody against a specific anti-blood group antigen" means a multi-strain, single-plant or recombinant antibody having a known specificity which recognizes and binds to a red blood cell carrying an antigen to which the antibody is labeled. Further preferably, the suspension of magnetic particles according to the present invention is characterized in that the anti-erythroblast blood type/phenotype antigen anti-system is selected from the following multi-plant or monoclonal antibodies (preferably monoclonal antibodies): anti-_ A, anti-3, anti-D, anti-c, anti-E, anti-c, anti-e, anti-K, anti-Fya, anti-Fyb, anti--, anti-throw, anti-S and anti- The -s antibody, or specifically against any other antibody that has a red blood cell gold/phenotype antigen that is present on the surface of the red blood cell. Again preferably, the suspension of magnetic particles according to the invention is characterized in that the magnetic particles are selected from the group consisting of superparamagnetic particles. Further preferably, the suspension of magnetic particles according to the present invention is characterized in that the magnetic particles are selected from magnetic particles having a diameter between 7575 μm and 5 μm, the diameter of the magnetic particles being It is preferred between i microns and 3 microns and between i microns and 95288 11 201209413 2 microns. Immunodiagnosis, cell capture (ceU capture) or cell sorting techniques using magnetic particles have been widely described in the publication and are well known to those of ordinary skill in the art. Among these techniques, 'particularly, some techniques utilize functionalization of magnetic particles to impart reactive surface functional groups that are capable of reacting with antigens, antibodies, or broadly under appropriate conditions and in the presence of appropriate reagents. In other words, any protein or its organism (such as a glycoprotein or a fragment thereof) grafted onto these particles in a covalently bonded manner will be reacted; such functional groups may include, in particular, a group, an amine group, The base, toluene, epoxide or acid functional groups are most commonly used. There are also techniques that use passive adsorption of antigens to be immobilized on the particles, but are less preferred; these particles can be sufficiently processed to produce positively or negatively charged beads, positively or negatively charged These antigens are determined by the conditions under which the passive adsorption is carried out. Further preferably, the suspension of magnetic particles according to the invention is characterized in that the magnetic particles have been previously functionalized with a group selected from the group consisting of a thiol, an amine group, a trans group or a fluorene group. Further preferably, the suspension of magnetic particles according to the invention is characterized in that it has been pre-selected with a group selected from the group consisting of silk, amine, sulfhydryl or anthracene and has a molar equivalent of 20 micro equivalents ( The functionalization ratio between eq)/gram of beads and 350 micro equivalents per agram of beads is preferably between μ micro equivalents per gram of beads and 80 micro equivalents per gram of beads. . Again preferably, the suspension of magnetic particles according to the invention is characterized in that the 12 95288 201209413 magnetic particles have an average iron content between 30% and 50%, the average iron content being between 30% and 40% The interval is better. Again preferably, the suspension of magnetic particles according to the present invention is characterized by a coating. The concentration of AHG covering the magnetic beads is between 1 μg/mg particles and 70 μg/mg particles; The anti-IgM type AHG is preferably between 20 ± 5 μg / mg of particles; in the case of anti-IgG type AHG, it is preferably between 50 ± 10 μg / mg of particles. 1%与2. 5。 The concentration of the magnetic particles is in the range of 0.1% and 2. 5 Between %(w/v) is better between 2. 2% and 1.5% (w/v), and between 0.25% and 〇% (w/v). Preferably, 0.5%±0.25% (w/v) is preferred, and 〇3% (>/illusion is preferred. The magnetic particle suspension is characterized in that the surfactant is selected from nonionic a surfactant or detergent selected from a nonionic hydrophilic detergent such as Tween (1) 20, 40 or 80; a polyalkylene oxide (p〇1〇xamer) such as

Sy叩eronic PE/F68 ® or F127 is preferred. Further preferably, the suspension of magnetic particles according to the present invention is characterized in that the concentration of the surfactant is between %. 1% and 2.5% (m/v), in 〇. and 1% ( Preferably, m/v) is preferably G 5% ± Q · 15% (m/v), and 0.75% ± 0.25% (m/v) is preferred. Further preferably, the suspension of magnetic particles according to the present invention is characterized in that the magnetic particle system of the ruthenium or the like is suspended in an aqueous buffer solution containing bovine serum albumin (10); the concentration of the BSA is 〇. 05% and 7. 75% (w/v), preferably between 〇 and 〇. 5% (W/V), 〇. 5% (w/v) is preferred; the water is slow 95288 13 201209413 The flushing solution is preferably a physiological buffer such as phosphate buffered saline (〇·3M, pH 7.4). Surprisingly, the inventors have shown that the molar concentration of such a buffer (about twice the molar concentration of standard PBS) can provide significantly better results, especially in this case coated with AG and if needed It is also a storage aspect of a suspension of magnetic particles coated with an antibody (ie, a ready-to-use suspension for blood type determination/phenotyping assay). Further preferably, the suspension of the magnetic particles according to the present invention is characterized in that the magnetic particles are suspended at a concentration of 0.1% ± 0.25% (w / v) containing 〇. 1% (w / v) Bovine serum albumin (BSA) in phosphate buffered saline (pBS) (〇. 3M, pH 7.4). In some applications, especially when it is desired to increase sensitivity or speed of reaction (but not increase the non-specificity of the assay), a low ionic strength buffer or LISS (low ionic strength solution) can be used. Those of ordinary skill in the art will understand that the term "buffer" or "saline solution" refers to a buffer commonly used in cell biology, particularly immuno-hematology, to avoid red blood cell lysis. Such a buffer or solution has, for example, a physiological pH between 6.8 and 7.5 and a molar concentration adjusted to be close to 0.9% NaCl solution of molar concentration (close to 〇·15M of MaCl) Buffer. One example of such buffers is phosphate buffered saline (PBS) having a 7 to 7 4 value, which is well known to those of ordinary skill in the art. Since LISS buffers are well known in the art-free hematology to increase agglutination, the composition of such buffers will not be described herein. This slow

14 95288 S 201209413 Infusion is obtained, inter alia, from a supplier of a liquid-free hematology reagent (one of several is a LISS buffer having the following composition: 16 g (g) / liter of glycine, 0.03 M NaCl And 0·015M phosphate, PH 6. 7). In another aspect, the invention includes a red-cartridge assay/phenotype assay kit comprising a suspension of magnetic particles according to the invention, wherein the anti-erythroblast blood type/phenotype antigen is specifically resistant to a given system antigen. Preferably, the red blood cell blood type assay/phenotype assay kit comprises at least two distinct magnetic particle suspensions according to the invention, wherein the antibodies against the red blood cell type/phenotype antigen each specifically counteract different Given antigen. Preferably, the red blood cell blood type assay/phenotype assay kit comprises a mixture of at least two magnetic particle suspensions according to the invention, wherein the antibodies against the red blood cell blood type/phenotype antigen each specifically counteract different given Antigen, with each anti-system against the weak! The anti-* of the antigen, part of the D antigen and/or D antigen is preferably preferred. In another aspect, the invention relates to a blood group assay/phenotyping assay for red blood cells containing a biological sample of a red blood cell that must determine a blood type or phenotype, the method comprising the steps of: a) allowing the container to be processed A suspension of red blood cells of a blood type or phenotype assay is contacted with a suspension of magnetic particles according to the invention, or with antibodies of different specificities (especially against certain phenotypes such as D_variant antigen or weak D antigen) a step of contacting at least two mixtures of the suspension of the magnetic particles; b) a step of cultivating the mixture of the particle suspension obtained in the previous step and the red blood cells (which can be stirred); 15 95288 201209413施加 Applying a magnetic field to the volume using a magnet located below the outside of the container = pulling the magnetic particles toward the magnet, if necessary, combining the red blood cells with the singapore magnetic particles, «by, will be in step c The red blood cell solution obtained by pressing together is resuspended; n in Y or by any other suitable reading system to read the presence or absence of the contents, the accumulation of agglomerates It is indicated that the antibodies against the red blood cell blood type/phenotype antigens of the magnetic particles are specifically resistant to the use of the red blood cell agglutination reaction according to the present invention to determine the blood group antigen (10) in the method towel, the step of applying a magnetic field Preferably, the container is preferably made using a magnet positioned below the exterior of the container such that the magnetic particles fall vertically to the bottom of the container. In a preferred embodiment, the invention relates to a method according to the invention, characterized in that the red blood cell blood type test/phenotype test method is characterized in that after step d), if necessary, the step of step (4) can be followed by A small agglomerate that may form a low-intensity reaction and that can form larger agglomerates by further steps is collected. In the meantime, the present invention relates to a method in which the red blood cell blood type comparison/phenotype mosquito method according to the present invention is characterized in that the erythrocyte suspension of the blood type silk type test in the step (a) is performed. The concentration in the aqueous solution is between 〇.2 ()% and Q. 1% (v/v), between 0.3% and 〇. 75% (v/v) Preferably, 5% (v/v) is preferred. In an equally preferred embodiment, the invention relates to a method, a root

95288 S 16 201209413 The red blood cell blood type test/phenotype verification method of the present invention is characterized in that the aqueous solution is a low ionic strength solution buffer. In an equally preferred embodiment, the present invention relates to a method according to the present invention, characterized in that the red blood cell blood type test/phenotype verification method according to the present month is characterized in that, in step b), the cultivation system is carried out between 10 minutes and 35 minutes. During this period, the period is preferably between 15 minutes and 30 minutes, preferably 2 minutes; The step b) is carried out at a temperature between 2 Torr and 4 Torr, preferably between 30 C and 37 c, preferably 37 ° C. In a particularly preferred embodiment, the invention relates to a method, the root=the red blood cell blood type test/phenotype verification method of the invention is characterized in that, in step c, the magnet is a permanent magnet and its strength is 1〇〇. Between Gaussian and =00 Gauss' is preferably 12,000 Gauss; applying a magnetic field to: between 2 and 5 minutes and 10 minutes, which is preferably 4 to 6 minutes. It is preferred to apply a magnetic field at room temperature. Six Ports In the method according to the present invention, the application of the magnetic field is preferably accomplished by a permanent magnet located below the outside of the reaction volume. In an equally preferred embodiment, the present invention relates to a method according to the present invention. The m-blood-type mosquito/table listening method according to the present day is stirred in step d) between 3 and 2 minutes. During this period, the period is preferably 1 to 2 minutes. In an equally preferred embodiment, the invention relates to a method according to the invention, characterized in that the red A-ball blood determination/phenotype verification method is characterized in that in step a), a red blood cell suspension obtained from a sample containing red blood cells is obtained. It is carried out from the red blood cells 95288 17 201209413 obtained by sedimentation of the whole blood sample taken from the individual. In an equally preferred embodiment, the present invention relates to a method according to the present invention, characterized in that the red blood cell blood type test/phenotype verification method is characterized in that in the step a) the container is a microplate to have a round bottom hole. It is better. In an equally preferred embodiment, the invention relates to a method according to the invention, characterized in that the red blood cell blood type test/phenotype test method is characterized in that in step b) the 'stirring system uses a microplate stirrer at 500 to 12 The speed is between 〇〇 rpm, which is preferably between 9 950 and 950 rpm. In a particularly preferred embodiment, the present invention relates to a method for red blood cell blood type verification according to the present invention. The phenotypic assay method is characterized in that in step d) the 'stirring system is carried out in two stages using a microplate, initially mixing between 1 〇 and 15 at a speed of 9 〇 rpm between 9 〇〇 and 1,200 rpm. The second and the second stage are stirred at a speed of 7 〇〇 rpro for a period of between 1 minute and 10 seconds and 1 minute and 45 seconds, and the period is preferably 1 minute and 3 seconds. In an equally preferred embodiment, the invention relates to a method according to the invention, characterized in that the red blood cell type/phenotype verification method is characterized by: at the end of step d) 'at a speed between 300 rpm and 500 rpm Further stirring is further stirred for 45 seconds at a speed of 450 rpm for a period of between 10 seconds and 1 minute. In an equally preferred embodiment, the invention relates to a method in which the red blood cell ▲•type assay/phenotype assay method according to the invention is characterized in that, in step a), 35 microliters (/z 1 ) is 45 μl (40 μL is preferred) 0·5% (v/v) red blood cell suspension with 10 μl of 〇. 3% (w/v) the magnetic particle suspension 18 95288

S 201209413 The float contacts in this hole. A device or apparatus comprising a red blood cell blood type assay/phenotype assay' characterized by comprising a suspension according to the invention. Preferably, the apparatus or kit for the presence or absence of a blood group antigen on the surface of the red blood cell in the red blood cell agglutination sample comprises: a) a container containing the magnetic body defined by the invention a suspension of particles; 〇 at least one magnet or __ group of magnets, disposed below the outside of the device; spear + c) for the system | and, if necessary, d) a reader, It is possible to evaluate the presence or absence of red blood cell agglutination in each container. The invention also relates to a device characterized in that the reaction volume is such that the hole having the bottom of the (four) shape or the v shape is better. Again preferably, the method according to the invention is characterized in that the aqueous solution of magnetic particles is previously diluted with a buffer or a saline solution. According to the present invention, the method for determining the presence or absence of an antibody against blood in the blood by the red blood cells (4) is determined by the method of determining the presence or absence of antibodies against the blood of the blood. The δ 'reaction capacity|| is preferably a micro disk having a round or V-shaped bottom. The hole is preferred. The following examples and illustrations are intended to illustrate the invention and are not intended to limit the scope of the invention in any way. Example 1: Formation of an immunomagnetic macromolecular complex An immunomagnetic macromolecular complex (see Fig. 1) was produced in two steps. 95288 19 201209413 A) Step 1, or one-time coupling Step 1, ie one-time coupling, involves immobilizing anti-IgG type and anti-IgM type anti-human globulin on magnetic beads containing functional groups. These functional groups may be of different types such as an amine functional group (-NH2), a hydroxy functional group (-0H), a delayed functional group (-C0O(R) or a toluenesulfonate functional group. All such functional groups are well known in the art. The linkage between the antibody and the magnetic beads can be electrostatic, hydrophobic or covalent. The covalent attachment of the antibody to the magnetic beads is achieved by functional groups on the surface of the magnetic beads. The magnetic beads used are superparamagnetic and have a size of 1 to 3 microns. These magnetic beads are hydrophobic and can be functionalized with a thiol or toluene sulfhydryl group depending on the antibody to be coupled. Among the available beads are, for example, magnetic beads from a number of different suppliers such as ESTAP0R, Dynal or JSRMicro. Preferably, the magnetic beads used are hydrophobic beads having a size of from 1 to 1.2 μm from ESTAP0R, or 1.5 μm beads from JSR Micro. The chemical functional group which can be used for immobilizing IgG type or igM type AHG may be an anthracene sulfonyl group, a carboxyl group, a chaotic type or a hydroxy type, and in the case of an anti-IgG type ahg, it is preferred to use a toluene sulfhydryl functional group. In the case of anti-type ahg, a carboxyl functional group is preferred. The average iron ratio of the magnetic beads is about 45% in terms of carboxylated beads and about 3% in terms of the fluorene-based beads. The functionalization rate of the functional groups can vary. In the case of toluenesulfonyl type beads, the number of functional groups may vary from 4 Å micro equivalents per gram of beads to 80 micro equivalents per gram of beads, and the number of fluorenyl sulfonyl functional groups is 75 micro equivalents / Gem is preferred; it has a carboxyl group

For the beads of 20 95288 S 201209413, the number of functional groups can vary from 20 micro equivalents per gram of beads to f 350 micro equivalents per gram of beads, and the number of carboxyl functional groups is 2 〇 micro equivalents per gram of beads. good. The anti-IgG type or anti-IgM type anti-human globin coupled to the functionalized magnetic beads is 1 of the same type (丨30 丫卩6).

For the coupling of anti-IgG type antiglobulin, the number of toluenesulfonyl beads from 1 to 1.2 μm from estapor (product number: R〇1_24, the number of indomethacin-based functional groups is 75 micro equivalents) / gram beads), 1.5 micron toluenesulfonyl beads from JSR (product number: MS15 〇 / acesulfonyl) is preferred. For the coupling of anti-IgM type antiglobulin, it is preferred to use a 1-micron carboxyl bead from ESTAP0R (product number: em1_1〇0/4〇). The amount of carboxyl functional groups of these beads was 20 micro equivalents per gram of beads. The concentration of AHG coupled to the magnetic beads can vary from 20 micrograms per milligram of beads to 50 micrograms per milligram of beads, depending on the type of antiglobulin. The anti-丨 type anti-human globulin line was used at a concentration of 50 μg/mg beads, and the anti-IgM type anti-human globulin line was coupled at a concentration of 20 μg/mg of beads. B) Step 2, or the second coupling in the second stage or the second coupling in the formation of the immunomagnetic complex is carried out by using the supernatant of the hybridoma culture secreting monoclonal antibody to erythrocyte antigen The specific antibody is coupled to the AHG composition. This coupling is achieved by culturing a bead of A H G coupled with a concentrate of monoclonal antibodies. The concentrate contains various proteins produced by the hybridoma-secreting hybridoma during the culture period of Pei 21 95288 201209413, and the coupling process allows the concentrate to be purified immunologically. After the second coupling, AHG is completely saturated by the specific igG type or IgM type antibody (determined by the coupled AHG), and constitutes a ready-to-use macromolecular immunomagnetic reagent which can be used for erythrocyte blood group antigen and phenotypic antigen. The assay 'the erythrocyte antigen to be assayed depends on the specific antibody (anti-A, anti-B, anti-D, anti-Fya, anti-Fyb, anti-Jka, anti-Jkb, etc.) used. Various hybridoma culture supernatant concentrates containing monoclonal antibodies specific for red blood cells can be coupled to AHG. Other types of immunomagnetic complexes can also be produced by coupling to identify anti-species globulins from other species (e.g., from mice) that are specific for erythrocyte antigens. Thus, for example, anti-globulin is anti-IgG type or IgM type anti-mouse globulin and antibodies against blood type/phenotype antigens are produced by mice.

IgG or IgM. The secondary coupling concentration depends on the amount of antibody present in the culture concentrate and the type of antibody. This concentration can vary from 120 micrograms total protein per milligram of beads to 3 micrograms total protein per milligram of beads. The final solution or storage buffer containing beads coupled to AHG and specific antibodies consists of PBS containing 1% (m/v) BSA and Synper(R) nic PE/F68 nonionic detergent. Synperonic PE/F68 is also known as the hydrophilic nonionic surfactant of the polyalkylene oxide Synperonic F68, available from Sigma Aldrich under the product number 81112. The three-stage polymer consists of a 2 hydrophilic polyoxy-extension ethyl block surrounding a central hydrophobic portion 22 95288 201209413 portion, the central hydrophobic portion comprising a polyoxypropylene propyl block. §When using nanoparticles, detergents must be used to avoid natural agglomeration of these nanoparticles under physical and chemical conditions such as specific pH, ionic strength and temperature. It has been demonstrated that changes in the surface of the particles caused by adsorption of amphoteric nonionic macromolecules such as polyalkylene oxides contribute to non-aggregation of particles in the solution. When particles are placed in a buffer solution having a high ionic strength such as pBS, the nanoparticles tend to lose their colloidal stability and agglutinate. The addition of this surfactant in a buffer with high ionic strength preserves colloidal stability and avoids agglomeration of the particles. There are several types of nonionic surfactants such as Tween 2, 6 and 80 or Synperonic PE/F68 or F127. In the present invention, it is preferred to use Sy叩eronic PE/F68e in order to improve the colloidal stability of the beads in which various antibodies are coupled. The concentration of Synperonic must be greater than 〇·1% and less than 2.5%. The cell survival threshold under the agent is preferably 1% (m/v). The final concentration of the beads coupled to the probed antibody depends on the antibody used and is in the above storage buffer (ie containing 0.1% BSA and 1%).

In PBS of Synperonic PE/68, the concentration of the beads may vary from 0.1 ° / 〇 to 0.5 % (m / v). Example II: Determination of blood type or phenotype The blood sample collected in a test tube containing an EDTA or citrate anticoagulant determines whether an antigen to be explored on the individual's red blood cells exists. Prepare the concentration in a test tube or in a round bottom well of a microplate by diluting the micro-liters of red blood cells that are tightly pressed together with 2 ml of low-pressure 23 95288 201209413 sub-strength buffer (especially LISS buffer). 〇· Between 3% and 1%, preferably 5〇/〇 of the globular suspension. A total of 10 microliters of a solution of beads coupled with an antibody (AHG + antibody specific for the antigen to be assayed) was deposited in a round well, and then 40 μl of a 0.5% red blood cell suspension was added. The compounds are homogenized by placing the microdiscs on an automated microplate stirrer. Stirring can be carried out at a speed of from 9 rpm to 1,200 rpm (preferably at a speed of 1, rpm) for a second. Then, the microplate is incubated at 37 ° C for 15 minutes to 3 minutes, preferably for 20 minutes, so that the antibody binds to the antigen present on the red blood cells. At the end of the cultivation, the microplate is placed on a plate containing 96 electromagnetic magnets, and the magnets are precisely adjusted to be located under the respective holes of the microplate. If the antibody has been combined with the red blood cells, under the action of the magnetic field, The magnetic beads pull the red blood cells down to the bottom of the hole and promote their agglutination. The magnetization is continued at room temperature for a period of 2 minutes and 1 minute, preferably 4 minutes. ... then place the weighing disc on the lion's apparatus to suspend the non-aggregated red blood cells (red blood cells) that are negative for the screened antigen. The following agitation sequence allows the non-agglutinated red blood cells to resuspend and the red blood cell agglutination to be seen: first, after magnetization, the relatively strong agitation causes the red blood cells that are tightly pressed together to no longer adhere and leave the bottom of the hole. The agitation can be carried out for 1 second in a coffee to simmer, 2 rpm change (preferably 900 rpm). The second disturbance caused the red blood of the negative material to be inscribed: the field floated, so the non-aggregator entered the suspension. This money can be mixed in 65 () to _ chat changes (better coffee 95288

S 24 201209413 2 Line 1 minute 3Q seconds. At the end of the agitation, the non-agglomerated ball is knives + homogenous suspension, and the red blood cells strongly expressing the antigen form a dense agglomerate. For antigens that are less intense, the agglomerates formed are smaller and more dispersed when the L-beam is stirred, so it is necessary to: "re-collect" all the scattered small aggregates into the holes. The bottom forms a complete, well-defined read set. The "recollection" agitation is preferably carried out at a low speed between 30 Torr and 50 rpm, preferably at a low speed of 45 rpm for at least 1 second but less than 丨 minutes. This "recollection" agitation has no effect on negative red blood cells or strong agglutination. The microdisc is then read by the naked eye or by an automatic reader equipped with a camera. EXAMPLE III Manufacture of an immunomagnetic macromolecular complex of IgG type anti-D To make the complex, various anti-D specific for certain variants of the D antigen can be used. 'Anti-D, _IgG type anti-D, HM16 (Diagast) strain _IgG type anti-D, P3X35 (Diagast) strain _IgG type anti-D, P3X290 (Diagast) strain - IgG type anti-D, ESD1 (Alba Biosciences) strain - IgM type anti-D, P3X61 (Diagast) strain - IgM type anti-D, P3X21223B10 (Diagast) strain known each anti-D To be able to identify certain types of D antigen variants. Table d below depicts the D antigen variants identified by each anti-D used. 95288 25 201209413 Table 1: Anti-Din running strains 2 and 3 are also weak D m 111 _ m T-* :·:.:· Correct 3 .. face 35:; following w; speak: 丨·:· Η!ΐ···ι ud·'; 1 ·' -: l|.-::. DFR:! r - i, > r 1 · · ·> :l,!W:!y >u - 1 ,v ' ill m;. 坤imqi QAU4 丨·!丨, - ;Scare thief® Lai S 4 4 4 ♦ ♦ ♦ ♦ » f + + 峙4 4 + 4 味 4 ♦ ♦ 令 + + ♦ 4 1 1 1 1 !/· 1(/-1. 1 1 •A 1 1 1 Tooth 4 •4 4 ♦ ♦ + ♦ - + + 4 4 呤♦ 4 + + tf- f ♦ . 4· + NT ♦A 4 ♦ + 13⁄4 - + • f . • + * ♦ - ♦ ¥ ♦ 4 4 + A) One-time coupling of anti-IgG anti-human globulin to sulfonium sulfonate beads a. Preparation of buffer sodium phosphate Buffer 0. 1M, pH 7. 4 - Weighing 2. 62 grams of NaH2P〇4xH2〇-weighing 14. 42 grams of Na2HP〇4xH2〇-dissolved in distilled water, then adjusted to 1 liter in volume

Ammonium sulphate buffer (NH4) 2S 〇 4, 3M - Weighing 39. 64 gram (NH4) 2S 〇 4 - Dissolved in 100 ml of sodium phosphate buffer - pH adjusted with NaOH or HC1 containing 0.5% (w /v) BSA in PBS (pH 7.4) (saturated buffer) (pbs refers to phosphate buffered saline) - Add 1. 53 grams of NaCl to 80 ml of saline-laden saline (0.02 M, ρΗ7. 4) Weigh one 中·5 gram of BSA (BSA refers to bovine serum albumin) - Mix one with sodium phosphate buffer (0.02M, pH 7.4) to adjust the volume to 1 〇〇 ml 95288 26 201209413 PBS containing 0·1% (w/v) BSA (pH 7_ 4) - Adding 1. 53 g of NaCl to 80 ml of phosphate buffered saline (0.02 M, pH 7.4) - weighing 0. 1 gram of BSA - mixed - adjusted to 1 〇〇 ml with sodium phosphate buffer (0.01M, pH 7.4) b. Reagents and materials - beads: benzene sulfonyl ruthenium beads (Product No.: R01-24), 1% (1 mg beads = 10 μl) - AHG antibody: purified anti-1 gG type AHG; initial concentration: 1.8 pg/ml - buffer: PBS (pH 7. 4); PBS (0.3 M, pH 7.4); ammonium sulfate

Flue (NH4) 2S 〇 4 (3M); PBS containing 0.5% BSA (pH 7.4); PBS containing 〇 · 1% BSA (pH 7.4) - Material: Pipettes (Eppendorf tubes), Adjustable aspirator and corresponding suction tube, mixing wheel, magnet and glass test tube. c. One-time coupling operation procedure The following operation procedure is repeated four times. 二次 Four types of IgG type anti-D: HM16, P3x35, P3x290 and ESDI can be used for secondary coupling. To achieve this, 4x15 mg beads (i.e., 4 x 150 microliters) were coupled with AHG at a ratio of 50 micrograms AHG per milligram of beads. Coupling 'put 150 μl of 10% (ie 15 mg) of sulfonium sulfonate beads into pipettes (Eppendorf® tubes) 27 95288 201209413 - by using magnets to rinse the beads - will contain 150 microliters of beads The pipette is placed on the magnet - the supernatant is aspirated while the beads are being pulled down - 1000 microliters of sodium phosphate buffer is added - the beads are resuspended in sodium phosphate buffer to form a suspension - the pipette is placed On the magnet - aspirate the supernatant - resuspend the beads together with 416 microliters (ie 750 micrograms) of antibody - mix thoroughly by thirsty - add 200 microliters of (NH4)2S〇4 buffer (3M) - Mix thoroughly by vortexing - Place the pipette on the mixing wheel at room temperature for 24 hours. Agitate and saturate the coupling beads - Place the pipette on the magnet and pull down the beads - Aspirate the supernatant Liquid - Resuspend the beads in 1 ml of a saturated buffer (PBS containing 0.5% BSA (pH 7.4)) - place the pipette on the magnet and pull down the beads - aspirate the supernatant Liquid - Resuspend the beads in 1 ml of a saturated buffer (containing 0.5% BSA in PBS (pH 7.4)) - incubate the beads on a stirring wheel for 2 hours at room temperature - shift this Liquid tube Place on the magnet and pull down the beads - aspirate the supernatant and replace 1 mL of PBS containing 0.1% BSA with 28 95288 g 201209413 - completely resuspend the beads by vortexing for 5 to 10 seconds - this The pipette was placed on the magnet and the beads were pulled down - the supernatant was aspirated - the beads were resuspended in 1.5% BSA in 1.5 ml of PBS and the final concentration of the beads was 1%. Secondary coupling of specific antibody (anti-D) and anti-IgG anti-human globulin a. Preparation of buffer containing 0.1% (m/v) BSA and 〇.5% (m/v) Synperonic PE/ F68® PBS (pH 7.4) (where Synperonic PE/F68 ® is the trade name for ethylene oxide and propylene oxide copolymers from ICI Group) ~ Add 1. 53 grams of NaCl to 80 ml of sodium phosphate buffer In the solution (0. 02M, PH7.4), weighed 0.1 g of BSA and 0.5 g of Synperonic PE/F68 ~ added to the phosphate buffer prepared above ~ mixed ~ with sodium phosphate buffer (〇. 〇2M, ρΗ7. 4) Adjust the volume to 100 ml of PBS containing 0·l% (m/v) and i% (m/v) Synperonic PE/F68® (ΡΗ7.4)~1·53 g Add NaCl to 80 ml of sodium phosphate buffer (〇. 〇2Μ, ΡΗ7 4) Medium ~ Weighing 〇 · 1 gram of BSA and 1 gram of Synperonic PE / F68 ~ Add this to the phosphate buffer prepared above. Mix 29 95288 201209413 - Use sodium phosphate buffer (〇 · 02M ' pH7 4) Adjust the volume to 100 ml b. Reagents and materials - beads. Magnetic beads coupled with anti-IgG type AHG at a concentration of 1% (ι mg beads / 100 μl) - Antibody:

HM16 : anti-D monoclonal antibody ' IgG type, culture concentrate P3x35 : anti-D monoclonal antibody, IgG type, culture concentrate P3x290 . Anti-D monoclonal antibody, IgG type, culture crushing solution ESDI : anti-D monoclonal antibody 'IgG type, culture concentrate-buffer: PBS containing 0.1% BSA; containing 〇. 1% BSA and 〇.. 5% Synperonic ® PE/F68 PBS - Material: Adjustable aspirator and corresponding pipette, glass tube, pipette (Eppendorf® tubes) and magnet c. Preparation of IgG type anti-D antibody for coupling Unpurified anti-D antibody concentrate with 15 The milligrams of toluene-based bead coupling (ratio: 120 μg/mg beads), wherein the indole-based beads have been previously coupled with AHG. - HM16: Anti-D monoclonal antibody 'IgG type' was obtained from the culture concentrate, and the antibody was not purified. Initial concentration: 11. 5 g/L of total protein added: 1800 μg, ie 155 μl volume - P3x35: Anti-D monoclonal antibody 'IgG type, obtained from culture concentrate, unpurified antibody initial Concentration: 10. 5 g / liter of all proteins 95288 30 201209413 Addition amount: 1800 micrograms, ie the volume of Π〇 microliters - P3x290: anti-D monoclonal antibody 'IgG type, obtained from culture concentrate, without purification The initial concentration of the antibody: 14 g / liter of the total amount of protein added: 1800 micrograms, that is, the volume of 130 microliters - ESDI: anti-D monoclonal antibody 'IgG type, obtained from the initial concentration of the culture concentrate: 16. 7 grams / liters of total protein added: 1800 micrograms, that is, 108 microliters of volume d · operating procedures 1.5 ml of AHG-coupled beads (15 milligrams of beads per second coupling) directly with the above volume of resistance - D antibody concentrate was incubated together - pipette was placed on a stirrer for 2 hours at room temperature - at the end of the second coupling 'put the pipette over the magnet, pull the beads down and aspirate the supernatant - Then use the beads in 1 ml containing 〇. 1% BSA The PBS was rinsed once with PBS containing 0.1% BSA and 5% Synperonic F68. The beads were resuspended in 1. 1% BSA and 1% Synperonic PE/F68. Store in PBS and store at 4 °C. I%BSA和1%。 I) The immuno-magnetic agent is diluted with the final buffer after the coupling of each immuno-magnetic complex containing 0.1% BSA and 1%

The PBS of Synperonic PE/F68 was diluted to a concentration of 0.3 〇/〇. Example IV: Production of IgM-type immunomagnetic macromolecular complexes A) Primary coupling of anti-IgM type anti-human globulin and carboxyl beads (c〇〇H) a. Reagents and materials 31 95288 201209413 Table 2 Beads Carboxyl carboxyl beads, concentration 10% (1 mg beads = 10 μl), Product Code: EM1-100/40 Antibody anti-IgM type AHG Anti-IgM type AHG (purification) Initial concentration = 1.1 mg / ml buffer NA2HP〇4/NAH2P〇4 10 mM, pH=6 NA2HP〇4/NAH2P〇4 20 mM, Ph=7. 5 2mM HC1 PBS containing 0.5% BSA (pH 7.4) PBS containing 0.1% BSA (pH 7. 4) coupling agent E1X; C18 mg/ml) and NHS (6 mg/ml) solution solution for NA2HPO4/NAH2PO4 (10 mM p H=6), pipette adjustable aspirator and corresponding suction tube agitating wheel magnet Glass test tube b. Procedure The following procedure was performed in two replicates using two IgM type antibodies: P3x61 and P3x21223B10 for secondary coupling. To do this, 2x15 mg beads (i.e., 2 x 150 microliters) were coupled with anti-lgM type AHG at a ratio of 20 micrograms AHG to 1 milligram of beads. Coupling: - Place 150 microliters of carboxyl beads at a concentration of 1% (ie 15 mg beads) in a pipette by rinsing the beads with a magnet: - Place a pipette containing 150 microliters of beads On the magnet - aspirate the supernatant when the beads are pulled down - add 1000 μl of buffer: Na2HP〇4/NaH2P〇4 (1 mM, pH 6) - Resuspend the beads by vortexing - using Na2HP〇 4/NaH2P〇4 (i〇mM, PH6) Repeat the rinsing step - move the beads into a 5 ml glass tube

32 95288 S 201209413 - Aspirate all supernatant - add 2.5 ml of coupling solution (EDC and nhs in Na2HP〇4/NaH2P〇4 (10 mM, pH 6)) - Resuspend the beads by vortexing - The tube was placed on a stirrer for 15 minutes at room temperature - at the end of the incubation, the tube was placed on a magnet and the beads were pulled down - the supernatant was aspirated - the beads were resuspended in 1 ml of 2 mM HCl buffer - Place the tube on the magnet and pull down the beads - aspirate the supernatant - resuspend the beads in 890 μl of Na2HP〇4/NaH2P〇4 (20 mM, pH 7.5) and immediately add to be coupled Anti-IgM type AHG, ie add 300 micrograms of AHG (ie 272 microliters of AHG) per 15 milligrams of beads. _ vortex_ place the tube on a mixing wheel for 2 hours at room temperature - use Na2HP (VNaH2P〇4 (2 mM, ρΗ7·5) Rinse the beads 2 times and resuspend the beads in 1.5 ml of Na2HP〇4/NaH2P〇4 (20 mM, pH 7.5). Rinse and saturate the coupling beads: _ Place the tube on the magnet and pull down the beads - aspirate the supernatant - resuspend the beads in 1 ml of saturated buffer, ie PBS containing 5% BSA (10) 7 · 4) 33 95288 201209413 _ Tube placement The beads were pulled down on the iron - the supernatant was aspirated and replaced with 1 ml of PBS containing 1% BSA - the beads were completely resuspended by vortexing for 10 seconds - the tube was placed on a magnet and pulled down Beads - aspirate supernatant - resuspend the beads in 1.5 ml PBS containing 1% BSA, the final concentration of beads is 1% B · specific antibody (anti-D) and anti-igM type -Second coupling of human globulin a. Reagents and materials Table 3 Beads and anti-IgM type AHG coupled magnetic beads, concentration 1% (1 mg beads = 1 〇〇 microliters) Product Code: EM1-100/ 40 antibody P3x61 anti-D monoclonal antibody, anti-IgM type, culture concentrate P3x21223B10 anti-D monoclonal antibody, anti-IgM type, culture concentrate buffer containing 0.1% BSA in PBS containing 0.1% BSA and 0 5% Synperonic PE/F68 PBS material---- Adjustable aspirator and corresponding pipette glass tube pipette magnet b. Preparation of the antibody to be coupled Unpurified anti-D antibody concentrate 15 mg of carboxyl bead coupling (and the ratio is as follows), wherein the carboxyl bead has been previously coupled with anti-IgM type AHG " for antibody Ρ3χ61, 320 μg anti- Body/mg beads Addition amount: 4800 micrograms (ie 457 microliters volume)

34 95288 S 201209413 - For the antibody P3x21223B10, 2000 μg antibody / mg beads added amount: 30,000 μg (that is, a volume of 1.2 ml). c. Procedure 1 1. 5 ml of the coupled anti-^ (4) Magnetic beads of type AHG (15 mg beads per second coupling) were directly cultured together with the above-mentioned anti-caries antibody concentrate. Place the pipette on the stirring wheel for 2 hours at room temperature. At the end of the sub-coupling, place the pipette over the magnet, pull down the beads and aspirate the supernatant - rinse the beads 1 ml with 1% BSA2 PBS and then use 〇. UBSA and 1% Synperonic * Wash the PE/F68 PBS twice - resuspend the beads in 1.5 ml of PBS containing 1% BSA & 1% 叩 PE/F68 and store at 4 ° C C) After the reagents were diluted and coupled with their final buffer, each immunomagnetic complex was diluted with PBS containing 1% BSA and 1% ic PE/, and diluted to a concentration of 抗°P p secret. 1%。 For the _Dp3x21223, diluted to a concentration of 0.1%. Example V: Examination of the use of various immunohybrids in blood phenotypic assays to detect faint and/or truncated isoforms of the antigen ° 95288 35 201209413 a. Reagents and materials 0· 3% beads In PBS containing 0.1% BSA and 0.5% Synperonic PE/F68, magnetic beads + AHG + HM16 magnetic beads + AHG + P3 χ 35 magnetic beads + AHG + P3 x290 magnetic beads + AHG + ESDI buffer containing 0.1% BSA and 0. 5% Synperonic F68 PBS LISS material adjustable applicator and corresponding pipette glass tube pipette magnet material 1 Greiner microplate with 96® bottom holes, irradiated with Freelys Nano reader sample in the presence of anticoagulant Whole blood control group (EQC) tube (positive control group) collected under (EDTA) test tube ΑΒ _ (negative control group) b. Preparation of 0.5% red blood cell suspension - collected from the presence of anticoagulant Take 5 μl of red blood cells that are tightly pressed together in a test tube of blood - dilute the red balls that are tightly pressed together in a test tube containing 1 ml of Liss - by stirring the homogenized suspension c · test for each sample and each Type bead preparation ~ use coupling Shake the container containing the beads before the beads

'•In a round bottom hole of a 96-well microplate, deposit 10 μl of beads coated with anti-D'. Add 40 μl of 〇·5% red blood cell suspension~ Stir the microplate at 900 rpm for a total of 10 Seconds to homogenize the beads and red blood cell suspension ~ incubated at 37 ° C for a total of 20 minutes

36 95288 S 201209413 One microplate was placed on a plate containing 96 electromagnets at room temperature for a total of 4 minutes 'where each electromagnet was in direct contact with each well of the microplate—the microplate was placed on an automated blender at 900 rpm Stir at a speed of 10 seconds' then stir at 70 rpm for 1 minute and 3 seconds, and finally at 45 rpm for 45 seconds - observe the microplate by naked eye to determine positive or negative reaction, or place the microplate On the automatic reader. The positive reaction is characterized by the presence of agglutination at the bottom of the well and the production of a homogeneous suspension indicating a negative reaction. Example VI: Attenuation or variability of rhesus monkey j) (RhesUs D, RhD) phenotype assay EFS anesthetic (Establishse/nents Francais du Sang, method) using various immunomagnetic complexes specific for D antigen The RhD phenotype of the Wind Transfusion Service, which has previously been confirmed by phenotypic testing to have a weak or mutated Rhesus D (RhD) phenotype. The phenotype of red blood cells obtained from several donors known to have D antigen variants or weak D antigens was assayed using various anti-D immunomagnetic complexes. Patients who did not have D antigen (RhD negative) were similarly assayed using the same immunomagnetic complex to demonstrate the specificity of the assay. a. Red blood cells with weak D antigen (weak D) 3 donors showed type 1 weak d (DwI) 7 donors showed type 2 weak sputum (Dw2) 2 donors showed type 3 weak D (Dw3) (See Figure 3) 95288 37 201209413 Each anti-D complex recognizes a variety of weak d antigens, but the intensity of the reaction depends on each clone. For example, the reactivity of the complex coupled with anti-DP3x290 and the weak D antigen of type 2 is slightly lower than that of the weak D antigen of type 1 and type 3. However, all of the IgG-type anti-D antigen antibodies coupled in the immunomagnetic complex are capable of agglutinating red blood cells exhibiting weak D antigen. Even when the antigen is extremely weak, for example, the type 2 weak D antigen (Dw2), the obtained reaction is still strong. b. Red blood cells showing part of the D antigen. Four blood donors showed d VI antigen (DVI). One blood donor showed D HMI antigen (DHMI). One blood donor showed DFR antigen (DFR). One blood donor showed D0L antigen (D0L). One donor presented DNB antigen (DNB) (see Figure 4). Each anti-D complex differed in the identification of the d antigen. The identification depends on the specificity of the epitope of each strain (see the map of the various sites of the strains used). Therefore, the immunomagnetic complex coupled with anti-D ΗΜ16 and anti-D Ρ3χ61 could not recognize the DVI-type partial d antigen. However, it was observed that all of the IgG type anti-D that was coupled in the immunomagnetic complex were capable of agglutinating red blood cells exhibiting a variant of the D antigen. The intensity of the response depends on the ability of each strain and its recognition variant. These reactions were as expected. The epitope of the anti-D strain used (see Table 5). 38 95288 201209413 Table 5 Strain 2 and 3 into D. L--S·!, D〇L w·; .*it. *.»·:::::· fiL .DU> • r . ::1: a«v LOUBv » . «.i .Γ^·;,. :^ΠΛ; Miscellaneous « l Wa;i 1 1 1 Gorge LV - 1 CMU /v. .,····_··· ' Vi 'ocs:_ - QFR 5 .r !::·.·; ». ae V -1· »_r 5 -. ,-» ' rOAR1,. .--1 DHK; mvk ,0AU4 a ♦ 4 + 4 ¥ + f®w«ea: + good 4 4 + + . 4 + 4 f + V- - + 4 + 4 ♦ 呤. • + + rae” Η· ♦ ♦ ♦ ν· + ♦ + N" 'V* ' Γ • 4 + Li. 4 - 4 4 ♦ ♦ - + c. Specificity of the test Use the same immunomagnetic complex to test the negative donor who is sampled under EDTA anticoagulant. The specificity of the test. The negative control group showed the expected negative reaction, while the positive sputum group (dyang's raw sample and DVI part positive sample) showed a strong positive reaction (see the fifth figure [Simplified illustration] against the first picture: Graphical representation of agglutinated immunomagnetic macromolecular complexes. From medium to external: magnetic particles (or magnetic beads) Anti-, (or anti-, anti-type) anti-human protein (AHG) ' and in the periphery against red blood cell blood type / phenotype antigens 2A and 2B map: use positive samples (2nd 8th) and negative samples (2B Figure 3. Reactions/results obtained. 3 ® . Use of secret samples for reaction / 2 and 3) ^ 1 95288 39 201209413 Figure 5: Reactions/results obtained using negative and positive samples (D positive and Part VI, Part D). Figure 6: Coupling of magnetic beads with anti-IgG or IgM type AHG. Figure 7: Anti-globulin and IgG anti-erythrocytes using unpurified monoclonal antibody concentrate Secondary coupling of antibodies. Figure 8: Principle of red blood cell phenotype assay by agglutination using an immunomagnetic macromolecular complex of IgG type Figure 9: Anti-ball using unpurified monoclonal antibody concentrate Secondary coupling of protein to IgM type anti-erythrocyte antibody. Fig. 10: Principle of red blood cell phenotype determination by agglutination using IgM type immunomagnetic macromolecular complex. [Main component symbol description]

40 95288 S

Claims (1)

201209413 VII. Patent application scope: 1. A suspension of magnetic particles characterized in that the magnetic particles are coated with an anti-globulin (AG)/antibody complex on the surface, wherein the antibody specifically targets the red blood cell type/ Phenotypic antigen (this antibody is referred to as "antibody against red blood cell type/phenotype antigen"). 2. The suspension of magnetic particles according to claim 1, wherein the AG is anti-human globulin (ahg) and the antibody against the red ash gold/phenotype antigen is an antibody derived from human. 3. The suspension of magnetic particles according to claim 1 or 2, wherein the AG is an anti-IgG type or an anti-IgM type anti-globulin. 4. The suspension of magnetic particles according to claim 3, wherein when the AG is of the anti-igG type, the antibody against the red blood cell type/phenotype antigen is IgG type, and when ag is anti-1 In the gM type, the antibody against the erythrocyte blood group/phenotype antigen is of the IgM type. 5. The suspension of magnetic particles according to any one of the preceding claims, wherein the AG is saturated with an antibody against a red blood cell type/phenotype antigen. 6. The suspension of magnetic particles according to any one of the preceding claims, wherein the anti-erythroblast blood type/phenotype antigen anti-system is selected from the following multi-plant or monoclonal antibodies (by monoclonal antibodies) Good): anti-A, anti-B, anti-D anti-〇 anti-E, anti-c, anti--6, anti-_^, anti-_^&, anti-_1?, anti-Jka, anti-shame, Anti-s and anti-s antibodies' or antibodies specifically against any other red blood cell blood type/phenotype antigen to be detected on the surface of the red sphere. · 95288 1 201209413 7·If you apply for the suspension of the magnetic particles of the 1st to 6th articles, the magnetic particles are selected from the superparamagnetic particles. 8. The suspension of magnetic particles according to the above paragraph, wherein the magnetic particles are selected from magnetic particles having a diameter between 0.75 micrometers and 5 micrometers, the diameter being A preference between micrometers and 3 micrometers is preferred between 1 micrometer and 2 micrometers. 9. A suspension of magnetic particles as described in paragraphs VIII to 8, wherein the magnetic particles have been previously functionalized with a group selected from the group consisting of a carboxyl group, an amine group, a hydroxyl group or a fluorene sulfonyl group. 10. The suspension of magnetic particles according to claim 9, wherein the magnetic particles are previously deuterated and functionalized with a group selected from a carboxyl group, an amine group, a hydroxyl group or a fluorene sulfonyl group. It is a magnetic particle between 2 microequivalents/gram of beads and 35 microequivalents per gram of beads (preferably between 20 micro equivalents/gram of beads and 8 microequivalents per gram of beads). The suspension of magnetic particles according to any one of claims 1 to 1 wherein the magnetic particles are magnetic particles having an average iron content between 30% and 4%. 12. The suspension of magnetic particles as described in any one of claims 1 to 11, wherein the concentration of AHG covering the magnetic beads is between 10 μg/mg of particles and 70 μg/mg of particles; In the case of anti-IgM type AHG, it is preferable to use between 20 ± 5 μg / mg of particles, and in the case of anti-IgG type AHG, it is preferable to use between 50 ± 10 μg / mg of particles. The suspension of magnetic particles according to any one of claims 1 to 12, wherein the magnetic particles are suspended in an aqueous solution containing a surfactant, 95288 S 201209413, the concentration of the specific particles Between 1% and 2.5% (w/v), preferably between 0. 25% and 1.5% (w/v), 〇. 5°/〇±〇 25%(w/v) is more preferred, preferably 0.3% (w/v). The suspension of magnetic particles according to claim 13, wherein the surfactant is selected from a nonionic surfactant or a detergent selected from a nonionic hydrophilic detergent such as Tween ( Tween®) 2〇, 40 or 80' polypyrazine (p〇iy〇xamer) such as Synperonic® PE/F68 or F127 is preferred; the concentration of the surfactant is 〇. 1% and 2. 5% (w/v) is preferably 7. 75% ± 0.25% (w/v). 15. The suspension of magnetic particles according to any one of claims 1 to 14, wherein the magnetic particles are suspended in an aqueous buffer solution containing bovine serum albumin (BSA); the concentration of the BSA 5%(w/v) between 0. 05% and 0. 75% (w/v) is preferably between 0. 1% and 〇. 5% (w/v). ) is better. 16. The suspension of magnetic particles of any one of claims 1 to 15 wherein the magnetic particles are suspended in a saline buffer, preferably suspended in a phosphate buffered saline solution. (〇. , PH7. 4). The suspension of magnetic particles as described in any one of claims 1 to 16, wherein the magnetic particles are suspended in a concentration of 0.1% ± 0.25% (w/v). Q(w/v) bovine serum albumin (No. SA) and 1% (w/v) of Sy叩eronic ® PE/F68 phosphate buffered saline (PBS) (0·3M ' ρΗ7· 4 )in. 18. A red blood cell blood type test/phenotype test kit comprising a suspension of magnetic particles according to any one of claims 1 to 17, wherein the 3 95288 201209413 is resistant to red blood cell type/phenotype antigen The system specifically targets a given antigen. 19. A red blood cell blood type test/phenotype test kit comprising at least two separate magnetic particle suspensions according to any one of claims 1 to 17, wherein the anti-erythrocyte blood type/phenotype antigen Each line of antibodies specifically targets a different given antigen. 20. A kit for use in a red blood cell blood type assay/phenotype assay, comprising a mixture of at least one of the magnetic particle suspensions of any one of claims 1 to 17 wherein the confrontation Each of the antibodies of the erythrocyte/phenotype sputum is specifically directed against a different given antigen, and each of the antibodies is preferably calibrated with an antigen selected from the group consisting of a weak D antigen, a partial D antigen, and a /stD antigen. 21. A device or apparatus for use in a red blood cell type* assay/phenotype assay, characterized by comprising a suspension of magnetic particles as described in any one of the above-mentioned claims. 22. A method for assaying/defining a red blood cell blood type, wherein a biological sample containing or phenotypically determined red blood cells is used, the method comprising the following steps: 2 suspending a red blood cell to be blood type or phenotype in a container The method of contacting the suspension of the magnetic particles according to any one of the items 1 to 17 or the step of contacting with at least two of the objects; the mixed iron of the suspension of μ & applying a magnetic field to the volume b a step of incubating, if necessary, by placing C) on the outside of the container to attract the magnetic particles, and if necessary, fixing the red blood cells to the magnetic particles; d) Re-suspending the red blood cells that are tightly pressed together in step c) by agitation; e) reading the presence or absence of agglomerates in the container with the naked eye and/or by any other suitable reading system The presence of agglomerates indicates the presence of an antigen that is specifically resistant to antibodies against the erythrocyte blood group/phenotype antigen on the magnetic particles. 23. The method of erythrocyte blood type assay/phenotypic assay according to claim 22, wherein, if necessary, after step d), further stirring may be performed, and the recollection may be formed with a low degree of reaction and may be further Stir to form a small agglomerate of larger agglomerates. 24. The method of claim 22, wherein the suspension of red blood cells to be subjected to blood type or phenotypic determination in step a) is a suspension suspended in an aqueous solution at a concentration of 0. Between 20% and 0.75% (v/v), preferably between 0.3% and 0.5% (v/v). The method of any one of claims 22 to 24, wherein the aqueous solution is a low intensity ion buffer. 26. The method of any one of claims 22 to 25, wherein the step b) (cultivation) is carried out between 10 minutes and 35 minutes, between 15 minutes and 30 minutes. Preferably, it is preferably 20 minutes; preferably step b) is carried out at a temperature between 20 ° C and 40 ° C, preferably between 30 ° C and 37 ° C, and 37 ° C is preferred. 27. The method of any one of claims 22 to 26, wherein in 5 C 288 201209413, in step C), the magnet is a permanent magnet having a strength of 1 〇, 〇〇〇 Gauss and 14, Between 1000 Gauss, 12 000 Gauss is preferred; the application time to the magnetic field of the container is 2.5 minutes to 1 minute, preferably 4 minutes to 6 minutes; It is better. 28. The method of claim 22, wherein the method of claim 22 is in any one of the claims. In step )), the mixing is performed for 3 to 2 minutes and 2 minutes and 3 seconds for 1 to 2 minutes. The method of any one of claims 22 to 27, wherein in step a) 'the red blood cell suspension obtained from the red blood cell-containing sample, the = is obtained after sedimentation of the whole blood sample taken from the individual It is carried out by red blood cells that are pressed together closely. The method of any one of claims 22 to 28, wherein in the step a) the container is a micro-disc, preferably a round-bottomed hole. 31. The method of claim 30, wherein in step b), the 'scrambler uses a microplate to perform at a speed between 500 and _rpm. The interval is better. The method of claim 3, wherein in the step d), the mixing system is carried out in two steps using a microplate, the first step being between 1 and 200 rpm, preferably g. 〇〇 rpm speed is mixed 1 〇 private and the second step is to disturb the 1 minute 1 〇 shift and 1 minute 45 seconds at 7 rpm, which is 1 minute 30 seconds is better. The method of any one of claims 22 to 28, wherein at the end of step d) at 95288 S 201209413, the mixture is further stirred at a speed between 300 rpm and 500 rpm for a period of between 10 seconds and 45 seconds. It is preferably further stirred at a speed of 450 rpm for 15 seconds. 5% (w), in the step a), in the step a), from 35 microliters to 45 microliters (preferably 40 microliters) of 0.5% (w /v) The red blood cell suspension is contacted with 10 microliters of 3% (w/v) of the magnetic particle suspension in the well. 35. A device or kit for determining the presence of a blood group antigen on a surface of a red blood cell in a sample by agglutination of red blood cells, characterized in that the device or kit comprises: a) a container containing, as claimed in claim 1 a suspension of magnetic particles as defined in any one of 17; b) at least one magnet or group of magnets disposed below the outside of the container; c) a stirring system for the container; Suitably, d) a reader that can assess the presence of red blood cell agglutination in each container. 36. The device of claim 35, wherein the reaction vessel is a microplate, preferably a hole having a round or V-shaped bottom. 7 95288