Universal detection plate for blood type and antiglobulin tests and application of universal detection plate

Abstract

The invention discloses a universal detection plate for blood type and antihuman globulin tests and application of the universal detection plate. The universal detection plate can be suitable for detection of all blood group antigens (including ABO blood group positive typing and reverse typing) and anti-human globulin, is good in detection effect and high in sensitivity, can well detect weak positive samples, and can present different degrees of agglutination strength. And the detection method based on the universal detection plate is simple and convenient to operate (4 * 96 tests per 10 minutes), clear in result, clear in negative and positive boundary and easy to interpret. The kit is simple in structure, convenient to operate, capable of avoiding tedious operation such as conventional anti-human globulin test washing and the like, capable of improving detection efficiency, more suitable for large-scale sample detection, free of missing detection of system blood group antibodies such as KEL and the like and capable of meeting the requirements for clinical rapid cross matching of blood and the like.

Description

Technical Field

[0001] This invention relates to the field of medical testing and medical device technology, and in particular to a universal test strip for blood typing and antiglobulin testing and its application. Background Technology

[0002] Blood typing microplate technology is an important detection method developed based on ELISA and microplate agglutination methods, and its earliest origins can be traced back to 1898. Modern microplate coating technology mainly involves fixing specific antibodies (such as anti-A, anti-B, anti-D, etc.) at specific locations on a microplate through drying or adsorption, and then identifying blood types through antigen-antibody reactions.

[0003] The "Capture Solid Phase Technology" developed by Immucor in the United States is currently the mainstream international approach, primarily used for antibody screening and identification in transfusion medicine. Its core advantage lies in the use of pre-coated solid-phase reaction plates, combined with IgG-specific reagents, significantly improving detection sensitivity and specificity. Furthermore, it simplifies result interpretation and reduces errors through intuitive color changes (after adding Capture LISS). This technology is easy to operate, eliminating the cumbersome steps of liquid chromatography and precise liquid aspiration required in traditional methods, shortening detection time, and flexibly adapting to manual, semi-automatic, and fully automated equipment (such as...). or This allows for single-sample or batch testing. Other commonly used methods include Diagast's solid-phase coating technology, which utilizes solid-phase adsorption to firmly immobilize highly specific monoclonal antibodies (for detecting erythrocyte antigens) or erythrocytes with known antigens (for detecting serum antibodies) onto carriers such as microplates or gel cards. This technology automates and standardizes blood typing (e.g., ABO / Rh) and antibody detection, offering advantages such as high sensitivity, high specificity, and good stability, and is often considered a representative method with relatively accurate detection results in this field. China introduced this technology in 2003 for the development of blood typing plates and has since developed various improved techniques such as solid-phase agglutination and magnetic bead methods.

[0004] However, the existing technology has many problems, such as: (1) Insufficient detection flexibility: the existing microplate uses a fixed combination of antibodies, which cannot be dynamically adjusted according to the detection needs, which can easily lead to duplicate detection or missed detection; (2) Inconsistent methodology: blood typing (direct agglutination method) and anti-human globulin test (indirect method) need to be performed separately, resulting in low detection efficiency; (3) High operational complexity: anti-human globulin test requires the addition of an isolation solution, making the process cumbersome; (4) Throughput limitation: the interpretation of results by traditional methods depends on manual observation, making it difficult to achieve high-throughput detection.

[0005] Therefore, there is an urgent need to develop a new coating technology that can integrate different detection methods, improve detection flexibility, and simplify operation procedures to meet the clinical testing needs for efficient and accurate blood typing. Summary of the Invention

[0006] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of this invention is to provide a universal detection strip for blood typing and antiglobulin testing and its application. Based on the universal detection strip of this invention, rapid determination of all blood group antigens (including ABO blood typing and reverse typing) and antiglobulins can be achieved with high sensitivity, effectively detecting even weakly positive samples and exhibiting varying degrees of agglutination intensity. Furthermore, no additional washing or other operations are required during the detection process, resulting in high detection efficiency and suitability for large-volume sample testing. It can cover antibody detection for multiple blood group systems such as ABO, Rh, and KEL. Moreover, due to its unaffected by antibody titer, it will not cause missed detections when used with blood group systems prone to false negatives, such as KEL, thus meeting the requirements for rapid crossmatching in clinical settings.

[0007] In a first aspect, the present invention provides a U-shaped microporous plate, wherein the U-shaped microporous plate is a U-shaped microporous plate that has been surface-treated with calcium chloride.

[0008] In some embodiments of the present invention, anti-erythrocyte membrane antibodies are immobilized in the pores of the U-shaped microplate.

[0009] In some embodiments of the present invention, the anti-erythrocyte membrane antibody includes at least one of anti-erythrocyte (RBC) monoclonal antibody and anti-erythrocyte (RBC) polyclonal antibody.

[0010] In some embodiments of the present invention, the calcium chloride solution also contains glycine.

[0011] In some embodiments of the present invention, the final concentration of glycine in the calcium chloride solution is 15-20 g / L.

[0012] In some embodiments of the present invention, the final concentration of glycine in the calcium chloride solution is 18 g / L.

[0013] In some embodiments of the present invention, the material of the U-shaped microporous plate includes at least one of polystyrene, polypropylene, polyethylene, polyethylene terephthalate, or polyethylene chloride.

[0014] In some embodiments of the present invention, the U-shaped microporous plate is made of polystyrene.

[0015] In some embodiments of the present invention, the surface treatment involves immersing the U-shaped microporous plate in a calcium chloride solution for 18-30 hours.

[0016] In some embodiments of the present invention, the concentration of the calcium chloride solution is 1.5-3M.

[0017] In some embodiments of the present invention, the concentration of the calcium chloride solution is 2M.

[0018] In this invention, based on surface treatment with a high-concentration calcium chloride solution, uniform 6-9 micrometer-sized micropores are formed at the bottom of the micropores in the U-shaped microplate, which can accommodate single red blood cells. Therefore, after red blood cells are added and coated with anti-red blood cell membrane antibodies, the presence of these micropores and the anti-red blood cell membrane antibodies increases the difficulty for single red blood cells to reach the bottom of the micropores. Only under specific centrifugation conditions can single red blood cells reach the bottom of the micropores under appropriate centrifugal force, thus forming a smooth and complete red blood cell loop at the bottom of the micropores. When red blood cells agglutinate with the corresponding antibodies, the agglutinated red blood cells form a network structure in the micropores and are captured by the antibody-coated red blood cell membrane. Centrifugation cannot move the agglutinated red blood cells, which have formed a network in the micropores, to the bottom of the micropores; instead, they form a flat layer within the micropores.

[0019] The presence or absence of a flat layer of red blood cells can be observed with the naked eye to determine the reaction between red blood cell antigens and corresponding antibodies, thereby determining the blood type.

[0020] A second aspect of the present invention provides a method for preparing the U-shaped microplate described above, comprising the following steps:

[0021] The U-shaped microplate was pretreated with calcium chloride solution, dried, and then coated with anti-erythrocyte membrane antibody into the wells. Blocking solution was added to block the microplate, thus obtaining the U-shaped microplate.

[0022] In some embodiments of the present invention, the concentration of the calcium chloride is as defined above.

[0023] In some embodiments of the present invention, the concentration of the calcium chloride is 2M.

[0024] In some embodiments of the present invention, the anti-erythrocyte membrane antibody is as defined above.

[0025] In some embodiments of the present invention, a coating buffer is also added during coating.

[0026] In some embodiments of the present invention, the coating buffer contains: disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, and potassium chloride (i.e., phosphate buffer); or, the coating buffer is at least one of carbonate buffer (containing sodium bicarbonate or disodium bicarbonate) or borate buffer (containing boric acid and sodium borate).

[0027] In some embodiments of the present invention, the coating buffer is composed of: disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, and potassium chloride.

[0028] In some embodiments of the present invention, the coating buffer also contains an adjuvant.

[0029] In some embodiments of the present invention, the excipients include at least one of preservatives, antibacterial agents, and stabilizers.

[0030] In some embodiments of the present invention, the adjuvant is a preservative.

[0031] In some embodiments of the present invention, the preservative includes sodium azide.

[0032] In some embodiments of the present invention, the final concentration of the anti-erythrocyte membrane antibody in the coating buffer is 0.25-6 μg / mL.

[0033] In some embodiments of the present invention, the final concentration of the anti-erythrocyte membrane antibody in the coating buffer is 4 μg / mL.

[0034] In some embodiments of the present invention, the amount of coating liquid used is 100-125 μL / well.

[0035] In some embodiments of the present invention, the amount of coating liquid used is 110 μL / well.

[0036] In some embodiments of the present invention, the sealing solution contains at least one of sodium caseinate, bovine serum albumin, and whey protein.

[0037] In some embodiments of the present invention, the sealing solution contains sodium caseinate and bovine serum albumin (BSA).

[0038] In some embodiments of the present invention, the blocking solution contains 1-10 g / L of sodium caseinate and 5-20 g / L of BSA, based on the final concentration.

[0039] In some embodiments of the present invention, the blocking solution contains 5 g / L of sodium caseinate and 12 g / L of BSA, based on the final concentration.

[0040] In some embodiments of the present invention, the amount of sealing liquid used is 150-175 μL / well.

[0041] In some embodiments of the present invention, the amount of sealing liquid used is 160 μL / well.

[0042] In some embodiments of the present invention, the sealing temperature is 18-26°C.

[0043] In some embodiments of the present invention, the sealing temperature is 22°C.

[0044] In some embodiments of the present invention, the sealing time is 20-30 hours.

[0045] In some embodiments of the present invention, the sealing time is 25 hours.

[0046] In some embodiments of the present invention, the sealing liquid also contains sucrose.

[0047] In some embodiments of the present invention, the sealing liquid further contains a solvent.

[0048] In some embodiments of the present invention, the solvent includes phosphate buffer.

[0049] In some embodiments of the present invention, the preparation method specifically includes:

[0050] Immerse the U-shaped microplate in 1.5-3M calcium chloride for 18-30 hours, dry the U-shaped microplate, mix the coating buffer and anti-erythrocyte membrane antibody as the coating solution to coat the U-shaped microplate, wash to remove the solution, add blocking solution to block, and dry to obtain the U-shaped microplate.

[0051] A third aspect of the present invention provides the application of the U-shaped microplate described above in blood typing and / or anti-immunoglobulin detection.

[0052] In some embodiments of the present invention, the blood typing includes, but is not limited to, at least one of: ABO blood typing positive / reverse typing, Rh blood group system antigen detection, MNS blood group system detection, KEL blood group system detection, LEVIS blood group system detection, KIDD blood group system detection, DUFFY blood group system detection, PIPK blood group system detection, and Lutheran blood group system detection.

[0053] In some embodiments of the present invention, the antiglobulin detection includes, but is not limited to, at least one of: direct antiglobulin test, accidental antibody screening, and crossmatch test.

[0054] A fourth aspect of the present invention provides a method for detecting blood type, comprising the following steps:

[0055] Add blood type antibody reagent or standard red blood cells to the U-shaped microplate described above, then add the blood sample to be tested, centrifuge at 1000-1200 rpm for 1-5 min, and determine the blood type based on the red blood cell agglutination.

[0056] In some embodiments of the present invention, the amount of blood sample added is 50-70 μL / microwell.

[0057] In some embodiments of the present invention, the amount of blood sample added is 50-60 μL / microwell.

[0058] In some embodiments of the present invention, the amount of blood sample added is 50 μL / microwell.

[0059] In some embodiments of the present invention, the blood sample to be tested includes red blood cell antigens or antibodies in serum / plasma.

[0060] In some embodiments of the present invention, when performing positive typological testing, the blood to be tested includes 0.3%-1.0% of positively typified red blood cells, added in an amount of 50-70 μL / microwell.

[0061] In some embodiments of the present invention, the volume of the blood type antibody reagent added is 15-45 μL / microwell.

[0062] In some embodiments of the present invention, the standard red blood cells are reverse-typed red blood cells.

[0063] In some embodiments of the present invention, the standard red blood cells are reverse-typed red blood cells with a concentration of 0.3%-1.0%, and when performing reverse typing, the volume of the blood sample to be tested is 50-70 μL / microwell.

[0064] In some embodiments of the present invention, the standard red blood cells include type A, B, or O reverse-typed red blood cells. Of course, those skilled in the art can select other reverse-typed red blood cells based on detection needs. Changing the type of reverse-typed red blood cells does not affect the detection effect of the method. Therefore, the standard red blood cells include, but are not limited to, type A, B, or O reverse-typed red blood cells.

[0065] In some embodiments of the present invention, the blood sample to be tested also includes a weakly positive sample of red blood cell antigens or antibodies in plasma.

[0066] In this invention, the “weakly positive blood sample” refers to a blood sample that is difficult to detect or cannot be detected by conventional rapid testing methods, but can be detected by complex laboratory testing, for a specific test item, where the content of the target analyte is too low or the presence of factors makes it difficult to detect.

[0067] In some embodiments of the present invention, blood type is determined based on the negative / positive results of the wells containing the corresponding blood type antibodies or standard red blood cells. This determination can be based on the general criteria for judging agglutination reactions in prior art blood typing tests.

[0068] In some embodiments of the present invention, when red blood cells agglutinate due to reaction with the corresponding antibody, after centrifugation, the agglutinated red blood cells cannot move due to mutual resistance and form a red blood cell layer, which is a positive reaction; non-agglutinated red blood cells will reach the bottom of the plate by the action of centrifugal force and form a red blood cell aggregate at the bottom of the reaction plate, which is a negative reaction.

[0069] In some embodiments of the present invention, the blood sample to be tested may be from a human or a non-human mammal.

[0070] In some embodiments of the present invention, the non-human mammals include: rodents, canines, felines, rabbits, horses, and primates.

[0071] In some embodiments of the present invention, the rodents include rats, mice, and guinea pigs.

[0072] In some embodiments of the invention, the primates include monkeys and apes.

[0073] In some embodiments of the present invention, the blood sample to be tested is from a human.

[0074] A fifth aspect of the present invention provides a method for detecting antiglobulins, the method comprising:

[0075] (1) Direct antiglobulin test, the steps are as follows:

[0076] Add 0.3%-1.0% of sample red blood cell suspension to the U-shaped microplate of claim 1, then add anti-human globulin reagent, centrifuge at 1000-1200 rpm for 1-5 min, and determine whether the red blood cells to be tested are sensitized by complement or IgG antibodies based on the red blood cell agglutination.

[0077] (2) Accidental antibody screening, the steps are as follows:

[0078] Add 50-70 μL / well of the serum / plasma to be tested and 0.3%-1.0% standard red blood cell suspension to the U-shaped microplate of claim 1, incubate for 15 min-30 min, add anti-human globulin reagent, centrifuge at 1000-1200 rpm for 1-5 min, and determine whether the blood sample contains unexpected antibodies based on the red blood cell agglutination.

[0079] (3) Crossmatch test, the steps are as follows:

[0080] Add 50-70 μL / well of patient serum / plasma and 0.3%-1.0% concentration of donor sample red blood cell suspension to the U-shaped microplate of claim 1; and add 50-70 μL / well of donor serum / plasma and 0.3%-1.0% concentration of patient sample red blood cell suspension. Incubate for 15 min-30 min respectively, then add anti-human globulin reagent, centrifuge at 1000-1200 rpm for 1-5 min, and determine whether the donor and patient blood are compatible based on the red blood cell agglutination of the two groups.

[0081] In some embodiments of the present invention, the definition of the standard red blood cells is the same as that defined above.

[0082] In some embodiments of the present invention, the anti-human globulin reagent includes at least one of anti-IgG antibody and / or anti-C3d antibody.

[0083] In some embodiments of the present invention, the source of the blood sample to be tested is limited as defined above.

[0084] A sixth aspect of the present invention provides a test kit or test apparatus comprising the U-shaped microplate described above and a test adjuvant.

[0085] In some embodiments of the present invention, the detection adjuvant includes at least one of the following: blood typing analysis diluent, physiological saline, sample release agent, standard reverse typing red blood cells, irregular antibody screening red blood cell reagent, blood typing antibody reagent, or anti-human globulin reagent.

[0086] In some embodiments of the present invention, the blood type antibodies, standard red blood cells, and anti-human globulin reagents are defined as described above.

[0087] The beneficial effects of this invention are:

[0088] This invention provides a universal microplate for blood typing and anti-human globulin detection. It requires only one type of microplate to detect different target analytes and can simultaneously perform multiple erythrocyte agglutination assays on the same plate. These include, but are not limited to, ABO positive and negative determination, anti-human globulin detection (direct antibodies, crossmatch antibody screening), Rh blood group system antigens (D, C, c, E, e), KEL blood group system antigens (K, k), MNS blood group system (M, N, S, s), and 43 other blood group systems. It effectively integrates technologies and improves the product's versatility.

[0089] Based on this universal detection plate, the present invention provides a method for rapid detection of blood type and anti-immunoglobulin. The method only requires centrifugation to obtain the test results within 5 minutes. The operation is simple and quick, with minimal human intervention, thus avoiding human error and improving the accuracy of the test.

[0090] Compared to traditional methods, the method in this invention eliminates the need for washing or introducing a separating solution, and also eliminates the need for additional antibody rehydration, thus reducing operational complexity and testing costs. Furthermore, it exhibits superior detection sensitivity, capable of identifying weakly positive samples and simultaneously performing forward and reverse ABO blood typing.

[0091] The method in this invention employs a discrimination method completely different from the prior art. By using the antibody at the bottom of the plate to capture agglutinated red blood cells while non-agglutinated red blood cells remain in the middle of the plate for identification, the reverse identification method can further distinguish weak positive samples.

[0092] The method in this invention can be applied to blood samples with excessively long storage periods, and the detection effect is consistent with that of fresh blood samples. It is not affected by fibrin or different anticoagulants, and does not miss the detection of systemic blood type antibodies such as KEL. It can meet the requirements of rapid crossmatching in clinical practice. Attached Figure Description

[0093] Figure 1 This represents the standard red blood cell agglutination status.

[0094] Figure 2 This represents the agglutination situation in actual testing.

[0095] Figure 3 This is the result of the uniformity test.

[0096] Figure 4 This is a diagram showing the detection effect of the method of the present invention on expired blood samples.

[0097] Figure 5 This is another detection effect diagram of the method of the present invention for expired blood samples.

[0098] Figure 6 The effect of the amount of blood sample added during reverse typing on blood typing detection. Detailed Implementation

[0099] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0100] Experimental materials

[0101] In the following examples, the sources of the relevant reagents used are shown in the table below.

[0102] Table 1. Information on the source of some reagents

[0103]

[0104] Example 1

[0105] In this embodiment, a universal test strip suitable for blood typing and antiglobulin assays is provided, the preparation method of which includes:

[0106] (1) Commercially available 96-well microplates made of U-shaped polystyrene (PS) were soaked in 2M calcium chloride containing 18g / L glycine for 18-30 hours (24 hours in this example). They were then washed three times with 0.01M PBS washing solution, spun dry, and dried at 37°C (humidity less than or equal to 30%).

[0107] (2) Coating of 96-well microplate:

[0108] Preparation of coating buffer: Weigh the following components of coating buffer according to the usage amounts: 14.507g disodium hydrogen phosphate dodecahydrate, 1.4823g sodium dihydrogen phosphate dihydrate and 0.22g potassium chloride, add purified water to make up to 1L, and then add sodium azide as a preservative at a rate of 1g / L.

[0109] Of course, it should be understood that, depending on the specific circumstances, the coating buffer in this invention can also be selected as 0.05 mol / L carbonate buffer (CB, pH 9.6), 0.01 mol / L phosphate buffer (1×PBS, pH 7.4) or 0.05 mol / L borate buffer (BB).

[0110] Add anti-erythrocyte membrane antibodies (different species of anti-erythrocyte membrane antibodies, including but not limited to mouse, sheep, and human anti-erythrocyte membrane antibodies, can be used depending on the actual application) to the prepared coating buffer. The antibody addition amount is 0.25-6 μg / mL, depending on the specific application. Add 75-125 μL of the coating buffer to each well of a 96-well microplate and allow it to adsorb at 2-8°C for 20-30 hours. Then discard the solution in the wells, add 200 μL of 0.01 M PBS as washing buffer, and shake and wash for 5 minutes. Discard the solution in the wells, pat dry, and repeat the washing and patting process 4 times.

[0111] (2) Closed:

[0112] Preparation of blocking solution: Add casein sodium to a final concentration of 1-10 g / L and BSA to a 0.01 mol / L PBS solution to obtain the blocking solution.

[0113] Add blocking solution to the dried microplate at a rate of 125-175 μL / well and seal at 18-26℃ for 20-30 hours. Discard the solution in the wells and invert the microplate after discarding the solution for 2 hours. Then place it in a dry plate chamber to dry completely, and the universal detection plate is obtained.

[0114] The obtained universal test plate was immediately sealed with a 96-well microplate sealing film, and then packaged with aluminum foil along with a desiccant for storage.

[0115] The universal blood typing and anti-human globulin detection microplate of this invention requires only one type of microplate to detect different target analytes, and can simultaneously perform multiple erythrocyte agglutination tests on the same plate. These include, but are not limited to, ABO positive and negative determination, anti-human globulin detection (direct antibodies, crossmatch antibody screening), Rh blood group system antigens (D, C, c, E, e), KEL blood group system antigens (K, k), MNS blood group system (M, N, S, s), and 43 other blood group systems. It effectively integrates technologies and improves the product's versatility.

[0116] Due to the diversity of blood group systems, it is impossible to exhaustively list all blood group system tests in the embodiments. Therefore, in the following embodiments, only the most common ABO and Rh blood group tests are used as representative examples. The usage and testing requirements of general blood group and anti-human ball testing microplates for other blood group systems are similar.

[0117] Example 2

[0118] This embodiment provides a blood typing method based on the universal testing plate in Embodiment 1 above. The specific steps are as follows:

[0119] For ABO blood type positive typing tests:

[0120] The red blood cell sample was diluted with physiological saline or isotonic saline (physiological saline was used in this example) to obtain a 0.8% red blood cell suspension. Then, the commercially available blood type antibody was added to the universal detection plate in Example 1 at a rate of 50 μL / well, followed by the 0.8% red blood cell suspension obtained in the above steps. The plate was centrifuged at 1200 rpm for 5 min, and the red blood cell agglutination of different sample red blood cells and different blood type antibody reagents in the microwells of the universal detection plate was observed to determine the blood type.

[0121] For ABO blood type reverse typing tests:

[0122] The standard reverse typing red blood cell reagent was diluted with physiological saline or isotonic saline (physiological saline was used in this example) to obtain a 0.8% red blood cell suspension. Then, sample plasma / serum was added to the universal detection plate in Example 1 at a rate of 50 μL / well, followed by the 0.8% red blood cell suspension obtained in the above steps. The plate was centrifuged at 1200 rpm for 5 min, and the red blood cell agglutination of different plasma samples and standard reverse typing red blood cells in the microwells of the universal detection plate was observed to determine the blood type.

[0123] The corresponding red blood cell agglutination patterns for different results are as follows: Figure 1 As shown.

[0124] In this method, a strong positive result is indicated when agglutinated red blood cells are evenly distributed throughout the microplate; a weak positive result is indicated when agglutinated cells are not completely distributed throughout the microplate, but only partially distributed at the bottom of the wells, but the binding area is larger than that of the negative control; and a negative result is indicated when red blood cells form regularly shaped red blood cell clusters at the bottom of the reaction wells. Compared to existing blood typing methods, the detection method in this embodiment can effectively detect weakly positive samples and exhibit varying degrees of agglutination intensity, demonstrating high sensitivity. Furthermore, compared to traditional methods, the detection method in this embodiment does not require washing or the introduction of a separating solution, nor does it require additional antibody rehydration, reducing operational difficulty and detection costs.

[0125] To further clarify the red blood cell agglutination situation, the inventors collected the following representative agglutination data (such as...). Figure 2 (As shown) and defined to effectively determine various agglomeration situations that may be encountered in actual testing.

[0126] Example 3

[0127] In this embodiment, in order to determine the effectiveness of the methods in the above embodiments, the inventors used the methods in the above embodiments and the test tube method (referring to the International Society of Blood Transfusion (ISBT) guidelines), which is currently the gold standard for blood typing, to perform blood typing on 36 known blood type samples containing different types of interfering substances (such as hemoglobin, lipids, bilirubin, etc.).

[0128] The results are shown in the table below.

[0129] Table 2. Blood typing results of the methods in the embodiments and the test tube method.

[0130]

[0131]

[0132] In the table, + indicates positive, numbers indicate intensity, and - indicates negative.

[0133] It can be observed that the detection results of the example method are basically consistent with those of the test tube method, but the example method is superior to the test tube method in terms of detection intensity, and the detection effect is more sensitive in the detection of some blood samples, without the false positive results of the test tube method. Moreover, commonly used interfering substances in clinical practice (such as hemoglobin, lipids, bilirubin, etc.) do not interfere with this methodology.

[0134] Example 4

[0135] In this embodiment, the inventors further conducted tests on several situations that are difficult to detect or have low detection sensitivity in the prior art, in order to verify the superiority of the method in the above embodiment compared with the prior art.

[0136] (1) Large-scale sample testing:

[0137] In this embodiment, 130 clinical blood samples were collected from the Bao'an District Central Blood Station in Shenzhen for testing, using the method described in Example 2 above. Blood group antibody-coated plates manufactured by Diagast were used as control reagents.

[0138] The results are shown in the table below.

[0139] Table 3. Blood typing results of the method in the examples and the solid-phase coating method.

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146] In the table, + / - indicates a weak positive result.

[0147] The results above show that the blood typing results obtained by the method in this embodiment are basically consistent with those obtained by the solid-phase coating method, which further confirms the accuracy of the method in this embodiment. However, in terms of detection efficiency, the method in this embodiment is more convenient and faster than the solid-phase coating method, and has higher detection efficiency and potency.

[0148] (2) Repeatability testing:

[0149] In this embodiment, the method described in Embodiment 2 is also used. Based on homologous blood samples, anti-A is used as a blood typing antibody to perform repeatable testing on the two universal test plates from Embodiment 1.

[0150] The results are as follows Figure 3 As shown.

[0151] It can be observed that the method of this embodiment has good repeatability, and the repeatability test results for homologous blood remain consistent with no significant differences. It has good batch-to-batch stability. Therefore, the method of this embodiment can be used for the detection of large-scale samples and can obtain reliable and trustworthy test results.

[0152] (3) Detection effect on weak positive samples:

[0153] In this embodiment, the methods described in the above embodiments and the Grifols ABO-Rh (Diagnostic Grifols, SA) microcolumn gel method were used to detect blood types in normal plasma (type O and type A plasma) and weakly positive plasma (type O and type A plasma).

[0154] The test results are shown in the table below.

[0155] Table 4. Detection efficacy of the methods and microcolumn gel method for weakly positive plasma in the examples.

[0156]

[0157] Based on the above results, it can be found that, for the same test sample, the method in this embodiment has a stronger detection sensitivity for weak positives than the existing microcolumn gel method, which is generally considered to have higher sensitivity. For example, for plasma samples numbered 3-4, 6-7, and 9-10 in Table 4, the method in this embodiment can show a more significant detection effect (reflected in intensity), thus avoiding the problem of misjudgment.

[0158] (4) Results of blood typing tests for Rh blood samples:

[0159] In this embodiment, 96 clinical Rh blood samples were collected from the Bao'an District Central Blood Station in Shenzhen for testing, using the method described in the above embodiment. A commercially available Rh blood sample testing card (purchased from Grifols DG Gel Rh Pheno (Diagnostic Grifols, SA), and the testing method was performed according to the instructions) was used as a control.

[0160] The test results are shown in the table below.

[0161] Table 5. Detection efficacy of the methods in the embodiments and commercially available Rh blood sample test cards on clinical Rh blood samples.

[0162]

[0163]

[0164]

[0165]

[0166] It can be observed that the results of the Rh blood typing test using the method in the examples are basically consistent with those of commercially available Rh blood sample test cards, further confirming the accuracy of the method in the examples. The inventors further verified the detection results using the microcolumn gel method, confirming that the detection results were correct.

[0167] (5) Accuracy of reverse typing red blood cell detection:

[0168] In this embodiment, 64 clinical blood samples with known blood types were collected. Reverse typing was performed using the methods described in the above embodiments and two commercially available reverse typing cell detection reagents (Shanghai Blood's human ABO reverse typing red blood cell suspension and Changchun Bode's ABO blood typing reverse typing kit (human red blood cells), with the detection method performed according to the instructions). The compatibility of the preservation solution in different standard reverse typing red blood cells was investigated.

[0169] Among them, blood samples of each type are tested in order of two spans of number (i.e., 1-2 are type A, 3-4 are type B, 5-6 are type O, 7-8 are type AB, 9-10 return to type A again, and so on in a cycle).

[0170] The results are shown in the table below.

[0171] Table 6 shows the detection efficacy of the methods in the embodiments and various commercially available Rh blood sample test cards on clinical Rh blood samples.

[0172]

[0173]

[0174]

[0175] It can be observed that the results of reverse typing blood typing using the methods in the examples are basically consistent with those using standard reverse typing reagents from different commercial manufacturers, further confirming the accuracy of the methods in the examples. However, since the methods in the examples are applicable to standard reverse typing reagents from different manufacturers, this further verifies that the detection principle of this methodology has strong reliability; it is based solely on whether agglutination occurs in the erythrocytes of the energy domain and is independent of other factors.

[0176] (6) Detection results for expired blood samples:

[0177] In this embodiment, 15 O-type clinical blood samples were collected and stored at 2-8℃ for more than 7 days beyond the clinical testing time, i.e., the storage time was as long as 15 days. The above-described method was used for testing. At the same time, fresh normal O-type clinical blood samples stored at 2-8℃ for 3 days were used as controls.

[0178] Each sample is tested in a column on a universal testing plate (one column per sample, tested on two universal testing plates). The specific setup is shown in the table below.

[0179] Table 7. Arrangement of blood samples in the universal test kit and their information.

[0180]

[0181] The results are as follows Figure 4 As shown.

[0182] It can be observed that the method described in the above embodiments can completely detect the blood type of the blood sample, and the detection effect is consistent with that of normal blood samples.

[0183] To further verify the accuracy of the results, the inventors used 64 clinical blood samples of type O (including expired and unexpired blood samples, arranged and with sample information as shown in the table below) for secondary verification. Each sample was placed in one well per column, with two replicates per column.

[0184] Table 8 shows the arrangement of blood samples in the universal testing kit and their information.

[0185]

[0186]

[0187] The results are as follows Figure 5 As shown.

[0188] It can be seen that the methods described in the above embodiments are indeed able to completely detect the blood type of the blood sample, and the detection accuracy is high.

[0189] Example 5

[0190] In this embodiment, the influence of different factors on the blood type detection method in the above embodiments was further investigated.

[0191] (1) The effect of different blood type antibody addition amounts on blood type detection:

[0192] In this embodiment, blood types A, B, and AB were used as test samples to investigate the effect of different amounts of added blood type antibodies on blood typing. The positive typing method based on the universal test plate is the same as in Embodiment 2, except that 25-75 μL of blood type antibodies were used respectively.

[0193] The results are shown in the table below.

[0194] Table 9. Effects of different blood type antibody dosages on blood type testing.

[0195]

[0196]

[0197] The above results demonstrate that the method in this embodiment of the invention has a good tolerance for different antibody loading volumes, thus exhibiting good adaptability to different loading volumes caused by equipment or human error. However, considering clinical sample loading habits, 50 μL / well for blood type antibodies is the optimal choice.

[0198] (2) The effect of the order of blood sample and antibody addition on blood typing:

[0199] In this embodiment, the impact of different blood sample and antibody addition orders on the detection results was examined. The positive typing method based on a universal detection plate was the same as in Embodiment 2, except that the order of blood sample and antibody addition was adjusted. Specifically:

[0200] The antibody-first, red blood cell suspension group was subjected to positive typing detection using the method described in Example 2, i.e., the antibody was added first, followed by the red blood cell suspension.

[0201] The order of adding blood sample and antibody in the positive typing detection method in Example 2 was reversed, i.e., the red blood cell suspension was added first and then the antibody was added.

[0202] The results are shown in the table below.

[0203] Table 10. Effects of different blood sample and antibody addition orders on blood typing.

[0204]

[0205] The above results indicate that there is no difference in the detection results obtained by adding blood type antibodies first and then adding red blood cell suspension versus adding red blood cell suspension first and then adding blood type antibodies. This is consistent with the expected results based on the detection principle of this method, which distinguishes between agglutinated and non-agglutinated red blood cells by centrifugation conditions, without being affected by other conditions.

[0206] (3) The effect of the amount of blood sample added during reverse typing on blood typing detection:

[0207] In this embodiment, AB type red blood cells were used as the test subject, and reverse typing blood typing was performed on blood samples with different added amounts, using type A reverse typing red blood cells and type B reverse typing red blood cells as standard red blood cells. The detection steps were the same as in Embodiment 2, except that the added amount of blood sample was set to 50 μL, 55 μL, and 60 μL, respectively (set as shown in the table below). Two wells / column were used for each sample.

[0208] Table 11. Blood distribution and blood information in the universal test kit.

[0209]

[0210] It can be found that 50-60 μL can make the test results stable and accurate. Based on the pursuit in this field of minimizing the amount of test samples, 50 μL of blood sample is preferred.

[0211] Example 6

[0212] In this embodiment, the applicability of the universal testing plate in Embodiment 1 and the blood typing method in Embodiment 2 in different blood typing systems was investigated. Using the traditional test tube method as a control, five relevant blood samples were randomly selected for testing.

[0213] The specific testing steps are as follows:

[0214] Red blood cells are obtained from the blood sample and then dissolved in isotonic saline to prepare a 0.7%–0.9% red blood cell suspension. 50 μL of blood typing antibody reagent (including MNS, KELL, DUFFY, KIDD, PIPK, and LEVIS blood typing system Fy) is added to each well of the universal testing plate. a 、Fy b 、Jk a 、Jk b Le a Le b ,P1,M,N,S,s,K,k,Di a Both Mur commercially available antigens and antibodies (all IgM type) and 50 μL of sample red blood cell suspension were placed in a centrifuge and centrifuged at 1200 rpm for 5 min. The results were then interpreted and recorded.

[0215] The traditional test tube method involves the following steps:

[0216] Red blood cells are obtained from the blood sample and then dissolved in isotonic saline to prepare a 2%-5% red blood cell suspension. In a clean test tube, add 50 μL of blood typing antibody reagent (including MNS, KELL, DUFFY, KIDD, PIPK, and LEVIS blood typing system Fy). a 、Fy b 、Jk a、Jk b Le a Le b ,P1,M,N,S,s,K,k,Di a Mix the Mur commercially available antigen (with IgM antibodies) and 50 μL of sample red blood cell suspension thoroughly, and centrifuge at 1000 rpm for 1 min (or equivalently at 900-1000 × g for 15 s). Gently shake the test tube to resuspend the red blood cells, and visually observe whether agglutination occurs at the bottom of the test tube.

[0217] The results are shown in the table below.

[0218] Table 12 Detection results of different blood group systems using the methods and test tube methods of the embodiments.

[0219]

[0220]

[0221] It can be seen that the detection results of the embodiment method are completely consistent with those of the test tube method, indicating that the embodiment method of the present invention has good detection effect on different blood group systems.

[0222] Example 7

[0223] In this embodiment, the detection efficacy of the universal test strip against anti-human globulin was tested, with several commercially available anti-human globulin test strips used as controls. These commercially available anti-human globulin test strips included: Changchun Boxun Biotechnology Anti-human Globulin Test Strip (batch number 20240401), Aikon Reagent Anti-human Globulin Test Strip (batch number 20240901), Diana Anti-human Globulin Test Strip (batch number 23307.01), and Dayamei Anti-human Globulin Test Strip (batch number 8820989802).

[0224] The detection procedure for anti-human globulin based on a universal detection plate is as follows: Take 50 μL of the plasma to be tested into the universal detection plate, add 50 μL of type O red blood cell suspension (type O standard red blood cells, 0.8%), incubate at 37℃ for 15 min, then add 50 μL of anti-human IgG, C3. Centrifuge at 1000 rpm for 5 min, and observe the results.

[0225] The detection method for commercially available anti-human globulin test cards should be performed according to the instructions for use.

[0226] Representative results of anti-human globulin detection are as follows: Figure 6 As shown in the table below, the specific detection results of this embodiment are as follows.

[0227] Table 13 Detection results of the general test strip and commercially available anti-human globulin test strip

[0228]

[0229] It can be seen that the universal detection plate in the embodiments of the present invention can basically achieve the detection accuracy of commercially available anti-human globulin detection cards. Therefore, it can be used for the detection of anti-human globulin. Moreover, compared with existing detection methods, the detection method of the present invention is simple and quick to operate, without too much human operation, avoiding human operation error and improving detection accuracy.

[0230] In summary, the universal detection plate of this invention can detect different target substances with only one type of microplate, and can simultaneously perform multiple tests of erythrocyte agglutination on the same plate.

[0231] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A U-shaped microporous plate, characterized in that, The U-shaped microporous plate is a U-shaped microporous plate that has undergone calcium chloride surface treatment; Anti-erythrocyte membrane antibodies are immobilized in the pores of the U-shaped microplate; The anti-erythrocyte membrane antibody includes at least one of anti-erythrocyte (RBC) monoclonal antibody and anti-erythrocyte (RBC) polyclonal antibody; The U-shaped microporous plate is made of at least one of polystyrene, polypropylene, polyethylene, polyethylene terephthalate, or polyethylene chloride.

2. The method for preparing the U-shaped microplate according to claim 1, comprising the following steps: The U-shaped microplate was pretreated with calcium chloride solution, dried, and then coated with anti-erythrocyte membrane antibody into the microwells. Blocking solution was added for blocking to obtain the U-shaped microplate. The concentration of calcium chloride is 1.5-3.0 molar concentration.

3. The preparation method according to claim 2, characterized in that, Coating buffer was also added during coating; The coating buffer contains at least one of the following: phosphate buffer, carbonate buffer, or borate buffer.

4. The preparation method according to claim 3, characterized in that, The coating buffer also contains excipients; The excipients include sodium azide.

5. The preparation method according to claim 2, characterized in that, The sealing solution contains at least one of sodium caseinate, bovine serum albumin, and whey protein.

6. The application of the U-shaped microplate of claim 1 in blood typing and / or anti-immunoglobulin detection; The blood typing test includes at least one of the following: ABO blood typing positive / reverse typing test, Rh blood group system antigen test, MNS blood group system test, KEL blood group system test, LEVIS blood group system test, KIDD blood group system test, DUFFY blood group system test, PIPK blood group system test, and Lutheran blood group system test. The antiglobulin test includes at least one of the following: direct antiglobulin test, accidental antibody screening, and crossmatch test.

7. A method for detecting blood type, comprising the following steps: Add blood type antibody reagent or standard red blood cells to the U-shaped microplate as described in claim 1, then add the blood sample to be tested, centrifuge at 1000-1200 rpm for 1-5 min, and determine the blood type based on the red blood cell agglutination. The blood sample to be tested includes red blood cell antigens, or antibodies in serum / plasma; or The blood sample to be tested contains 0.3%-1.0% positively typified red blood cells; The amount of positively typified erythrocytes added is 50-70 μL / microwell.

8. The detection method according to claim 7, characterized in that, The volume of the blood type antibody reagent added is 15-45 μL per microwell.

9. The detection method according to claim 7, characterized in that, The standard red blood cells are reverse-typed red blood cells with a concentration of 0.3%-1.0%; when adding standard red blood cells, the volume of the blood sample to be tested is 50-70 μL / microwell.

10. A method for detecting anti-human globulin, the detection method comprising: At least one of the following: direct antiglobulin detection method, accidental antibody screening method, and crossmatching test method; The unexpected antibody screening method includes the following steps: Add 50-70 μL / well of the serum / plasma to be tested and 0.3%-1.0% concentration of standard red blood cell suspension to the U-shaped microplate of claim 1, incubate for 15 min-30 min, add anti-human globulin reagent, centrifuge at 1000-1200 rpm for 1-5 min, and determine whether the blood sample to be tested contains unexpected antibodies based on the red blood cell agglutination. The direct anti-human globulin detection method includes the following steps: Add 0.3%-1.0% of sample red blood cell suspension to the U-shaped microplate of claim 1, then add anti-human globulin reagent, centrifuge at 1000-1200 rpm for 1-5 min, and determine whether the red blood cells to be tested are sensitized by complement or IgG antibodies based on the red blood cell agglutination. The crossmatch test method includes the following steps: Add 50-70 μL / well of patient serum / plasma and 0.3%-1.0% concentration of donor sample red blood cell suspension to the U-shaped microplate as described in claim 1; and add 50-70 μL / well of donor serum / plasma and 0.3%-1.0% concentration of patient sample red blood cell suspension. Incubate for 15 min-30 min respectively, then add anti-human globulin reagent, centrifuge at 1000-1200 rpm for 1-5 min, and determine whether the donor and patient blood are compatible based on the red blood cell agglutination of the two groups. The anti-human globulin reagent includes at least one of anti-IgG antibody and / or anti-C3d antibody.

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