Sample adding structure for anti-human globulin test kit, kit and detection method

Abstract

The invention provides a sample adding structure for an anti-human globulin test kit, which comprises at least one sample adding tube and at least one sample adding tube, the tube body is provided with a sample adding cavity; at least one through hole is formed in the bottom of the sample adding cavity; a base; the base is provided with a fixing structure which is used for fixing reaction holes coated with anti-human globulin at the bottom of the kit body. Through the design of the base, the sample adding structure body can be matched with a reaction hole of a conventional U-shaped reaction plate in the market. The sample adding pipe is used for providing an incubation space for red blood cells and a to-be-detected sample and preventing a reaction liquid from being diluted due to direct contact with an exclusion chromatography separation medium, so that the detected sample and a detection reagent can fully react in the sample adding pipe, the detection sensitivity of the kit is improved, and meanwhile, sample adding is facilitated; on the other hand, through centrifugation after incubation is completed, an exclusion chromatography separation medium is used for directly separating a red blood cell immune complex and a free irrelevant antibody, so that a tedious washing process is avoided.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a sample addition structure, a kit and a detection method for an anti-human globulin test kit. Background Art

[0002] Most IgG blood type antibodies in the human body are incomplete antibodies. IgG immunoglobulin antibody molecules are monomers with small molecular weight (150KD). Their Fab segments recognize and bind to corresponding antigens on the surface of red blood cell membranes, but do not cause red blood cell agglutination. The Fab segment of anti-human IgG immunoglobulin (AHG) recognizes the Fc segments of two adjacent IgG immunoglobulins on red blood cell membranes, thereby bridging and causing red blood cells that have been bound to incomplete antibodies to agglutinate. The agglutination strength generally increases with the increase in the amount of immunoglobulin bound to red blood cells. The test for detecting incomplete antibodies is called the anti-human globulin test, also known as the Coombs test (Anti-human globulin Test or Coombs Test). More than 40 years after the invention of the Coombs test, it has become one of the most important experimental techniques in clinically ensuring safe blood transfusion, diagnosing and preventing neonatal hemolytic disease, autoimmune hemolytic disease, drug-immune hemolytic disease, etc., as well as in blood type research. However, because the Coombs test procedure is complicated, requires repeated washing, and is time-consuming, for a long time, the Coombs test has only been used in confirmatory experiments on a few special case specimens and has never become a routine clinical test item.

[0003] In the routine antibody screening, identification and cross-matching of clinical blood type serology, it is required that incomplete antibody testing should be performed on recipients and donors with a history of blood transfusion, pregnancy, and transfusion of blood products. The Coombs test is the most clear and accurate method in theory, but due to its cumbersome operation and the inability to test multiple specimens at the same time, the test can only be used in the definitive test of a few specimens. For a long time, in larger hospitals and blood transfusion units in my country, routine clinical antibody testing can only be combined with a three-step test. That is, first a saline hemagglutination test, then a screening test with the addition of a hemagglutination-promoting reagent, and finally a definitive test on a few specimens. In more clinical hospitals, only saline hemagglutination tests or screening tests are used. In the 1960s, many immunolabeling techniques began to appear, such as RIA, immunofluorescence analysis, ELISA, etc. Among them, the immune labeling detection technology based on the anti-human globulin test principle, that is, the use of antigen-antibody, has been attempted many times for routine testing of red blood cell blood type serology. However, due to reasons such as the method being too sensitive, the experimental procedure being complicated, time-consuming and inconvenient, it has not been able to replace the traditional test tube Coombs test in clinical routine.

[0004] In the 1990s, scientists invented the microcolumn gel immune assay (MGIA). The anti-human globulin test was carried out in a microcolumn gel medium, overcoming the cumbersome washing and negative confirmation procedures of the traditional test tube method. It has the advantages of high sensitivity, easier results and long-term storage. It has been used in red blood cell blood typing tests in countries around the world. However, the shortcomings of MGIA technology in clinical application cannot be ignored: (1) MGIA uses gel particles to achieve no-washing. The fibrinogen present in the sample to be tested will form fibrin in the gel, hindering the sedimentation of red blood cells and floating in the gel to form a false positive. Old red blood cell fragments will also cause false positive results; (2) The essence of MGIA is an agglutination test, so it has low sensitivity and misses weakly reactive antibodies. Therefore, there is an urgent need for a new anti-human globulin detection technology that is simple to operate, accurate in detection, high in sensitivity and low in price. (3) The core raw material of MGIA, the separation medium gel particles, are mostly imported from abroad, which is expensive and difficult to control independently. Summary of the invention

[0005] In order to achieve the above objectives, the present invention is implemented through the following technical solutions.

[0006] The present invention provides a sample adding structure for an anti-human globulin test kit, comprising at least one sample adding tube, wherein the sample adding tube is a plastic part and comprises:

[0007] Tube body; the tube body is provided with a sample adding cavity for adding a sample to be tested or a sample to be tested and a detection reagent for an anti-human globulin test; the bottom of the sample adding cavity is provided with at least one through hole for passing liquid under centrifugal action;

[0008] A base, used to support the tube body;

[0009] The base is provided with a fixing structure for fixing in the reaction well coated with anti-human globulin at the bottom of the reagent box body.

[0010] Preferably, a plurality of convex ribs are evenly distributed on the outer peripheral side of the base for abutting against the inner surface of the reaction hole to achieve fixation.

[0011] Preferably, the bottom surface of the sample loading chamber is a concave curved surface.

[0012] Preferably, the number of the through holes is at least four; one of the through holes is arranged at the center of the bottom of the sample loading chamber; and the other through holes are distributed in a ring array around the center of the bottom of the sample loading chamber.

[0013] Preferably, the number of the sample adding tubes is eight or twelve in a row.

[0014] Preferably, two adjacent sample adding tubes are provided with a first connecting piece.

[0015] Preferably, the first connecting member is disposed laterally near the top of the sample adding tube for marking.

[0016] Preferably, two adjacent first connecting members are staggeredly arranged to be close to two sides of the sample adding tube respectively.

[0017] The second object of the present invention is to provide a kit, including a kit body, wherein the kit body includes:

[0018] The sample loading structure for the anti-human globulin test kit as described above;

[0019] The U-shaped reaction plate is provided with eight or twelve rows of reaction wells; the bottom of the reaction wells is coated with anti-human globulin; and the reaction wells are filled with exclusion chromatography separation medium;

[0020] The sample adding structure body is an eight-row or twelve-row sample adding tube structure, which is fixed to the U-shaped reaction plate through a base, and one of the sample adding tubes is fixed on one of the reaction holes.

[0021] The third object of the present invention is to provide a detection method for anti-human globulin test, using a kit as described above to detect incomplete antibodies, specifically comprising the following steps:

[0022] S1, coating anti-human globulin at the bottom of the reaction well;

[0023] S2, install the sample tube on the reaction hole;

[0024] S3, adding a size exclusion chromatography separation medium into the sample tube, and centrifuging to allow the size exclusion chromatography separation medium to enter the reaction well;

[0025] S4, adding the sample to be tested and the detection reagent to the sample tube, mixing or mixing and incubating;

[0026] S5. Centrifuge and observe the results.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention provides a sample loading structure for an anti-human globulin test kit. Through the design of the base, the sample loading structure body can be compatible with the reaction wells of a conventional U-shaped reaction plate on the market to form a test kit, which is easy to assemble. The sample loading tube is used to provide an incubation space for red blood cells and samples to be tested, and to prevent the reaction liquid from directly contacting with the exclusion chromatography separation medium and being diluted, so that the tested sample and the detection reagent can fully react in the sample loading tube, improve the detection sensitivity of the test kit, and facilitate sample loading; on the other hand, after the incubation is completed, the red blood cell immune complex and free irrelevant antibodies are directly separated by centrifugation using the exclusion chromatography separation medium, thereby avoiding a tedious washing process.

[0029] In a preferred embodiment, a plurality of convex ribs are provided on the outer peripheral side of the base to form the fixing structure, so that the sample loading structure body can be easily assembled with the reaction hole of the U-shaped reaction plate, and it is not easy to loosen and fall off during the subsequent centrifugation operation; in addition, there are gaps between adjacent convex ribs for air permeability, and combined with the design of the number and size of through holes, the detection reagent and the sample to be tested will not pass through the through holes during the incubation process in the sample loading chamber, and the liquid in the sample loading chamber can smoothly enter the reaction hole during the subsequent centrifugation process.

[0030] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. The specific implementation of the present invention is given in detail by the following embodiments and their accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0032] Figure 1 It is a schematic diagram of the three-dimensional structure of the sample adding structure body in one embodiment of the present invention;

[0033] Figure 2 This is a schematic diagram of the explosion structure of a test kit in one embodiment of the present invention;

[0034] Figure 3 Schematic diagram of the three-dimensional structure of a reagent kit in one embodiment of the present invention;

[0035] Figure 4 A top view of a sample loading structure body in another embodiment of the present invention;

[0036] Figure 5 It is a front view of a sample loading structure body in another embodiment of the present invention;

[0037] Figure 6 A front view of a U-shaped reaction plate in one embodiment of the present invention;

[0038] Figure 7 The schematic diagram of the indirect anti-human globulin test detection method of the present invention based on cross-matching blood in the embodiment;

[0039] Figure 8 The present invention is a flowchart of the steps of the detection method.

[0040] In the figure:

[0041] 1. Test kit body;

[0042] 100, sample adding structure body; 10, sample adding tube; 11, tube body; 111, sample adding cavity; 112, through hole; 12, base; 121, rib; 13, first connecting member; 14, second connecting member;

[0043] 200. U-shaped reaction plate; 20. reaction hole; 21. third connecting member; 22. support frame. DETAILED DESCRIPTION

[0044] The present invention is further described in detail below in conjunction with the accompanying drawings, and the aforementioned and other purposes, features, aspects and advantages of the present invention will become more obvious, so that those skilled in the art can implement it with reference to the text of the specification. In the accompanying drawings, for the sake of clarity, the shape and size can be enlarged, and the same reference numerals will be used in all figures to indicate the same or similar parts. In the following description, words such as center, thickness, height, length, front, back, rear, left, right, top, bottom, upper, lower, etc. are based on the orientation or position relationship shown in the accompanying drawings. In particular, "height" is equivalent to the size from top to bottom, "width" is equivalent to the size from left to right, and "depth" is equivalent to the size from front to back. These relative terms are for the convenience of explanation and are generally not intended to require specific orientation. Terms related to attachment, connection, etc. (for example, "connection" and "attachment") refer to the relationship that these structures are directly or indirectly fixed or attached to each other through intermediate structures, and movable or rigid attachment or relationship, unless otherwise explicitly stated.

[0045] The present invention is further described below in conjunction with the accompanying drawings and specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form a new embodiment.

[0046] In solid phase immunosorbent assay, a specific U-shaped reaction plate is required for the test; the usual U-shaped reaction plate consists of a strip and a frame, each strip consists of a number of reaction wells, and the strip is fixed to the frame through a certain structure. The existing anti-human globulin test is used for incomplete antibody detection, and the U-shaped reaction plate on the market is directly used for detection. After the red blood cell antigen is incubated and combined with the incomplete antibody, it needs to be washed to remove the free antibody, and then the anti-human globulin reagent is added or reacted with the anti-human globulin reagent coated at the bottom of the well to achieve detection. The washing is cumbersome and inefficient.

[0047] The present invention provides a sample loading structure for an anti-human globulin test kit, such as Figure 1 , Figure 2 , Figure 3 As shown, it includes a sample adding structure body 100, and the sample adding structure body includes a sample adding tube 10; the sample adding tube 10 is a plastic part, including:

[0048] Tube body 11; the tube body 11 is provided with a sample adding chamber 111 for adding a sample to be tested or a sample to be tested and a detection reagent for an anti-human globulin test; the bottom of the sample adding chamber 111 is provided with at least one through hole 112 for passing liquid under centrifugal action;

[0049] A base 12, used to support the tube body 11;

[0050] The base 12 is provided with a fixed structure for fixing the bottom of the reagent kit body 1 in the reaction well 20 coated with anti-human globulin. Specifically, the above-mentioned reaction well 20 is the reaction well structure on the conventional U-shaped reaction plate on the market. The U-shaped reaction plate on the market is a 96-well plate, including an eight-well structure with twelve holes or a twelve-well structure with eight holes. According to the design of the base 12, the sample tube 10 is commonly used in the reaction well 20 of the conventional U-shaped reaction plate on the market. When the anti-human globulin test is used for incomplete antibody detection, the bottom of the reaction well 20 is coated with anti-human globulin, and there is an exclusion chromatography separation medium in the hole. The sample loading chamber 111 is used to add the sample to be tested or the sample to be tested and the detection reagent. Among them, the exclusion chromatography separation medium is a separation medium that can separate the cellular components and non-cellular components in the object to be detected by centrifugation to remove non-specific interference, and realize the separation of the particulate antigen-antibody immune complex from plasma or serum. Therefore, the purpose of accurate and rapid detection can be achieved without washing during the detection process. When the substance added to the sample chamber 111 is a red blood cell antigen-antibody complex that has been sensitized in the human body, or the added sample material to be tested contains incomplete antibodies, it will form an antigen-antibody complex with the reagent red blood cells. Under the action of centrifugation, the red blood cell antigen-antibody complex passes through the through hole 112 and the exclusion chromatography separation medium in turn, enters the bottom of the reaction well 20, and binds to the anti-human globulin. The red blood cell antigen-antibody complex is combined with the anti-human globulin and is flattened at the bottom of the reaction well 20 to present a red blood cell flat layer; when the substance added to the sample chamber 111 is a red blood cell that has not been sensitized in the human body, or the added sample material to be tested does not contain incomplete antibodies, no red blood cell antigen-antibody complex is formed. Under the action of centrifugation, the red blood cells pass through the through hole 112 and the exclusion chromatography separation medium in turn, enter the bottom of the reaction well 20, and form a red blood cell buckle that aggregates at the bottom of the well. The results are interpreted by the different distribution and aggregation morphology of the red blood cells presented at the bottom of the reaction well 20. No washing is required during the detection process, and the operation is simple and efficient. By designing the sample tube 10, filling the reaction hole 20 with the exclusion chromatography separation medium, and separating the reaction space at the bottom of the reaction hole 20 from the exclusion chromatography separation medium in the reaction hole 20, on the one hand, the red blood cells in the sample tube 10 can be fully combined with the incomplete antibodies, while avoiding the direct contact between the red blood cells or antibodies and the exclusion chromatography separation medium, thereby improving the detection sensitivity; on the other hand, after the incubation is completed, the red blood cells and free antibodies are directly separated by centrifugation using the exclusion chromatography separation medium, thereby avoiding the cumbersome washing process. In addition, through the design of the base 12, the sample loading structure body 100 can be adapted to the reaction hole 20 of any U-shaped reaction plate 200 on the market, which is easy to assemble and convenient for loading.

[0051] Specifically, when the test is a direct anti-human globulin test, the red blood cells of the object to be tested are added to the sample chamber 111 as the test sample. If the red blood cells to be tested are sensitized by incomplete antibodies, there will be antigen-antibody complexes on the surface of the red blood cells. After centrifugation, the red blood cell antigen-antibody complexes pass through the through hole 112 and the exclusion chromatography separation medium and enter the bottom of the reaction well 20, and combine with the anti-human globulin at the bottom of the well, presenting a red blood cell layering phenomenon at the bottom of the reaction well 20, which is positive; when the red blood cell antigens to be tested are not sensitized by incomplete antibodies, after centrifugation, the red blood cells pass through the through hole 112 and the exclusion chromatography separation medium in turn and enter the bottom of the reaction well 20, forming a red blood cell button at the bottom of the reaction well 20, which is negative.

[0052] When the test is an indirect anti-globular test, such as Figure 7 As shown, the presence of incomplete antibodies in the serum or plasma (test sample) is detected by using screening cells or spectrum cells as a detection reagent, or the presence of incomplete antibodies in the red blood cells (test sample) is detected by using known incomplete antibodies as a detection reagent. Similarly, the detection reagent and the test sample are added to the sample loading chamber 111, incubated and centrifuged, and enter the reaction well 20, separated by the exclusion chromatography separation medium, and the antigen-antibody complex enters the reaction well and is captured by the anti-human globulin, and the test result is positive; otherwise, it is negative.

[0053] In one embodiment, the size exclusion chromatography separation medium includes a polysucrose-diatrizoate mixture and a secondary antibody solution, the specific gravity of the medium is 1.05-1.10, and the osmotic pressure is 280-350 mmol / L. The secondary antibody includes but is not limited to goat anti-human IgG, rabbit anti-human IgG, mouse anti-human IgG monoclonal antibody or chicken anti-human IgY antibody.

[0054] In one embodiment, if Figure 1As shown, a plurality of convex ribs 121 are evenly distributed on the outer peripheral side of the base 12 to form the fixing structure, which is used to abut against the inner surface of the reaction hole 20 to achieve fixation. Further, the sample tube 10 is an integrally formed structure. Further, after the base 12 is inserted into the open end of the reaction hole 20, the inner surface of the reaction hole 20 squeezes the convex ribs 121 so that the sample tube 10 and the reaction hole 20 are firmly assembled. Specifically, when there is no centrifugal operation, the liquid in the through hole 112 in the sample chamber 111 has a certain surface tension and will not pass through the through hole 112. The convex rib 121 is made of plastic material and has a certain elasticity. When the base 12 is inserted into the open end of the reaction hole 20, the convex rib 121 is squeezed so that the sample tube 10 and the reaction hole 20 are firmly assembled. In addition, there is a gap between adjacent convex ribs 121, which is breathable. After the sample tube 10 is installed on the reaction hole 20, the air inside the reaction hole 20 can circulate with the outside air, thereby ensuring that the atmospheric pressure in the sample chamber 111 is consistent with that in the reaction hole 20. When centrifugal operation is involved, the liquid in the sample chamber 111 can smoothly enter the reaction hole 20 through the through hole 112. That is, through the structural design of the base 12, the sample loading structure body 100 can be easily assembled with the reaction hole 20 of the U-shaped reaction plate 200, and is not easy to loosen and fall off during the subsequent centrifugation operation; in addition, combined with the design of the number and size of the through holes 112, the detection reagent and the sample to be tested will not pass through the through holes 112 during the incubation process in the sample loading chamber 111, and in the subsequent centrifugation process, the liquid in the sample loading chamber 111 can smoothly enter the reaction hole 20.

[0055] In one embodiment, the bottom surface of the sample loading chamber 111 is a concave curved surface, so that the liquid in the sample loading chamber 111 can pass through the through hole 112 smoothly during the centrifugal operation, and avoid cells being stuck in the angle due to the sharp angle between the two sides of the sample loading chamber 111. Furthermore, the outer contour of the through hole 112 is smooth, combined with the design of the ribs 121 on the base 12, so that the liquid in the sample loading chamber 111 will not pass through the through hole 112 during the incubation process, and the liquid can enter the reaction hole 20 during the subsequent centrifugation process, and no red blood cells will remain in the sample loading chamber 111, so as to reduce the impact of the red blood cells remaining in the sample loading chamber 111 on the detection repeatability and the impact on the accuracy of the later result imaging and the result interpretation by the software.

[0056] In one embodiment, the number of the through holes 112 is at least two, and the structural dimensions of the through holes 112 are consistent to control the liquid seepage effect at the bottom of the sample loading chamber 111 .

[0057] Furthermore, the number of the through holes 112 is at least four; one of the through holes 112 is disposed at the bottom center of the sample loading chamber 111; and the other through holes 112 are distributed in a circular array around the bottom center of the sample loading chamber 111. In one embodiment, the number of the through holes 112 is six, and the six through holes 112 are distributed in a shower-like manner, so that the liquid in the sample loading chamber 111 can be spread on the exclusion chromatography separation medium after passing through the through holes 112 to accelerate separation.

[0058] In one embodiment, if Figure 4 , Figure 5 As shown, the number of the sample tubes 10 is eight or twelve in a row, and one sample tube 10 is used to detect one sample. Figure 6 As shown, a U-shaped reaction plate 200 with eight parallel connections is used as an example for specific description, and the sample adding structure body 100 is designed as an eight-connected sample adding tube structure (i.e., the number of sample adding tubes 10 is eight) to match the U-shaped reaction plate 200. Furthermore, the U-shaped reaction plates on the market are usually eight-connected twelve-hole or twelve-connected eight-hole structures, and the sample adding structure body 100 is designed as an adaptive eight-connected twelve-hole or twelve-connected eight-hole structure to match conventional U-shaped reaction plates on the market.

[0059] Furthermore, if Figure 4 As shown, the two adjacent sample tubes 10 are provided with a first connector 13, which plays a connecting role. Furthermore, the first connector 13 is arranged horizontally near the top of the sample tube 10 for marking. The first connector 13 is arranged horizontally, and the upward side of the first connector 13 has a certain surface area, and is arranged near the top of the sample tube 10, so that the user can write a mark on the upper surface of the first connector 13 during the detection process to mark the corresponding sample tube 10. Furthermore, since the overall size of the sample adding structure body 100 is small, the surface area size of the first connector 13 is effective, and a marker is used to form the mark.

[0060] Furthermore, two adjacent first connecting members 13 are staggeredly arranged to be close to two sides of the sample adding tube 10 respectively, so as to visually distinguish the marks so as to prevent the user from confusing the adjacent sample adding tubes 10 .

[0061] In one embodiment, the first connecting member 13 is disposed transversely. Figure 5 As shown, two adjacent sample adding tubes 10 are provided with a second connecting member 14 , and the second connecting member 14 is vertically arranged to increase the connection firmness between the two adjacent sample adding tubes 10 .

[0062] The present invention also provides a kit, such as Figures 1 to 6 As shown, the kit body 1 includes:

[0063] The sample loading structure for the anti-human globulin test kit as described above is used for loading samples;

[0064] The U-shaped reaction plate 200 is provided with eight or twelve rows of reaction wells 20; the bottom of the reaction wells 20 is coated with anti-human globulin; the top of the anti-human globulin is filled with an exclusion chromatography separation medium; wherein the anti-human globulin includes anti-human IgG immunoglobulin and / or anti-human IgM immunoglobulin; when it is necessary to detect IgG incomplete antibodies, the bottom of the reaction well 20 is coated with anti-human IgG immunoglobulin; when it is necessary to detect IgM incomplete antibodies, the bottom of the reaction well 20 is coated with anti-human IgM immunoglobulin; when it is necessary to detect IgG incomplete antibodies and IgM incomplete antibodies, the bottom of the reaction well 20 is coated with both anti-human IgG immunoglobulin and anti-human IgM immunoglobulin;

[0065] The sample adding structure body 100 is a structure of eight or twelve rows of sample adding tubes 10, which are fixed to the U-shaped reaction plate 200 through a base, and one sample adding tube 10 is fixed to one reaction hole 20. The sample adding tube 10 and the reaction hole 20 are assembled to obtain a detection tube for detecting a sample.

[0066] Specifically, when assembling the reagent kit body 1, firstly, anti-human globulin is coated on the bottom of the corresponding reaction well 20, and then the sample loading structure body 100 is installed on the U-shaped reaction plate, and the size exclusion chromatography separation medium is added to the corresponding sample loading cavity 111, and the size exclusion chromatography separation medium is thrown into the reaction well 20 by centrifugation. During the detection, the sample to be tested and the detection reagent are added to the sample loading cavity 111, incubated, centrifuged, and the phenomenon at the bottom of the reaction well 20 is observed for interpretation.

[0067] In one embodiment, two adjacent U-shaped reaction plates 200 are connected by a third connecting member 21; a support frame 22 is provided on both sides of the U-shaped reaction plate 200, so that the user can hold the U-shaped reaction plate 200 and the matching imaging instrument can support the U-shaped reaction plate 200 during subsequent imaging.

[0068] The present invention also provides a detection method for anti-human globulin test, such as Figure 7 , Figure 8 As shown, using the above-mentioned kit or the above-mentioned detection card to detect incomplete antibodies specifically includes the following steps:

[0069] S1, coating the bottom of the reaction well 20 with anti-human globulin; wherein the anti-human globulin is filled with an exclusion chromatography separation medium; wherein the anti-human globulin includes anti-human IgG immunoglobulin and / or anti-human IgM immunoglobulin;

[0070] S2, installing the sample tube 10 on the reaction hole 20; in one embodiment, the sample tube 10 is inserted into the open end of the reaction hole 20 through the base 12;

[0071] S3, adding a size exclusion chromatography separation medium to the sample tube 10, and centrifuging to allow the size exclusion chromatography separation medium to enter the reaction well 20;

[0072] S4, adding the sample to be tested and the detection reagent to the sample tube 10, and mixing or mixing and incubating;

[0073] S5. Centrifuge and observe the results.

[0074] In order to illustrate the invention of this article, the following is described with specific examples. It should be understood that these examples are only for illustrative purposes and should not be understood as limiting the present invention in any way.

[0075] Example 1

[0076] Direct antiglobulin test

[0077] (1) Preparation of the kit

[0078] Dilute the anti-human globulin to 20 μg / mL with coating buffer (carbonate buffer, pH 9.6), add it to the reaction well 20 in the 96-well U-shaped reaction plate (100 μL / well), and place it in a 4°C refrigerator for 12 to 16 hours. Take the microplate out of 4°C, discard the supernatant, add 200 μL / well of 2wt% BSA blocking solution, and block it at 37°C for 2 hours. Discard the supernatant, wash it 5 times with washing solution (0.01M phosphate buffer with pH 7.2 + 0.05wt% Tween-20), and the amount of washing solution used for each wash is 250 μL / well. Finally, completely remove the liquid in the reaction well 20, control it on absorbent paper, and dry it at 37°C. Assemble and fix the upper sample loading structure body 100 of the U-shaped reaction plate coated with anti-human globulin, put it into an aluminum foil bag, add desiccant, vacuum seal it, and store it at 4°C for use.

[0079] (2) Detection

[0080] Take out 100 of the test kit body according to the detection amount and mark it. The remaining 100 of the test kit body should be put back into the ziplock bag and sealed and stored at 2-8°C. Use 0.9% sodium chloride to dilute the red blood cells to be tested into a 0.15% cell suspension for use. Add 150μL of exclusion chromatography separation medium to the sample chamber 111, centrifuge at 1000rpm for 10s, and centrifuge the exclusion chromatography separation medium into the reaction well 20. Add 75μL of 0.9wt% sodium chloride solution to the sample chamber 111, then add 50μL of red blood cell suspension and mix well. Use a TD-3A centrifuge, centrifuge at 1600rpm for 3min, and centrifuge at 1400rpm for 5min. Determine the test results.

[0081] Example 2

[0082] Indirect antiglobulin test for cross matching

[0083] (1) Preparation of the kit

[0084] Dilute the anti-human globulin to 20 μg / mL with coating buffer (carbonate buffer, pH 9.6), add it to the reaction well 20 in the 96-well U-shaped reaction plate (100 μL / well), and place it in a 4°C refrigerator for 12 to 16 hours. Take the microplate out of 4°C, discard the supernatant, add 200 μL / well of blocking solution, and block at 37°C for 2 hours. Discard the supernatant, wash 5 times with washing solution (0.01M phosphate buffer with a pH of 7.2 + 0.05wt% Tween-20), and the amount of washing solution used for each wash is 250 μL / well. Finally, completely remove the liquid in the reaction well 20, control it on absorbent paper, and dry it at 37°C. Assemble and fix the upper sample loading structure body 100 of the qualified U-shaped reaction plate, put it into an aluminum foil bag, add a desiccant, vacuum seal it, and store it at 4°C for use.

[0085] (2) Detection

[0086] Separation of red blood cells and plasma / serum from the donor and recipient.

[0087] Use 0.9wt% sodium chloride to prepare 0.1% to 0.15% red blood cell suspension for the red blood cells of the recipient and the donor, respectively. Take the test kit body 1 to be tested, add the exclusion chromatography separation medium (150μL / well) to the sample chamber 111, and centrifuge at 1000rpm (centrifugal force 131g) for 10s. One sample tube 10 corresponds to one reaction well 20 to form a test tube. Each sample to be tested requires two wells, i.e. two test tubes. Add 50μL / well of 0.9wt% sodium chloride solution to the sample chamber 111. Add 25μL of the recipient's plasma / serum and 50μL of the donor's red blood cell suspension to the first well (primary side); add 25μL of the donor's plasma / serum and 50μL of the recipient's red blood cell suspension to the second well (secondary side), and incubate at 37℃ for 15min. Immediately use a dedicated centrifuge to centrifuge at 1600 rpm (centrifugal force 335 g) for 3 minutes, and at 1400 rpm (centrifugal force 256 g) for 5 minutes, take out, and judge the result with the naked eye.

[0088] Example 3

[0089] Indirect antiglobulin test for screening of irregular antibodies

[0090] (1) Preparation of the kit

[0091] Dilute the anti-human globulin to 20 μg / mL with coating buffer (carbonate buffer, pH 9.6), add it to the reaction well 20 in the 96-well U-shaped reaction plate (100 μL / well), and place it in a 4°C refrigerator for 12 to 16 hours. Take the microplate out of 4°C, discard the supernatant, add 200 μL / well of blocking solution, and block at 37°C for 2 hours. Discard the supernatant, wash 5 times with washing solution (0.01M phosphate buffer with a pH of 7.2 + 0.05wt% Tween-20), and the amount of washing solution used for each wash is 250 μL / well. Finally, completely remove the liquid in the reaction well 20, control it on absorbent paper, and dry it at 37°C. Assemble and fix the upper sample loading structure body 100 of the qualified U-shaped reaction plate, put it into an aluminum foil bag, add a desiccant, vacuum seal it, and store it at 4°C for use.

[0092] (2) Detection

[0093] Take out the test kit body according to the detection amount and mark it. The remaining test kit body should be put back into the ziplock bag and sealed and stored at 2-8°C. Dilute the irregular antibody detection reagent (screening cells) with 0.9wt% sodium chloride solution to a 0.15% cell suspension for use. Add 150μL of exclusion chromatography separation medium to the corresponding sample chamber 111, centrifuge at 1000rpm for 10s, and centrifuge the exclusion chromatography separation medium into the reaction well 20. Add 50μL of diluent to the sample chamber 111, then add 25μL of the plasma or serum sample to be tested, and then add 50μL of red blood cell suspension and mix well. Incubate at 37°C for 15min. Centrifuge at 1600rpm for 3min and 1400rpm for 5min. Determine the test results.

[0094] Example 4

[0095] Indirect anti-human globulin test for identification of irregular antibodies

[0096] (1) Preparation of the kit

[0097] Dilute the anti-human globulin to 20 μg / mL with coating buffer (carbonate buffer, pH 9.6), add it to the reaction well 20 in the 96-well U-shaped reaction plate (100 μL / well), and place it in a 4°C refrigerator for 12 to 16 hours. Take the microplate out of 4°C, discard the supernatant, add 200 μL / well of blocking solution, and block at 37°C for 2 hours. Discard the supernatant, wash 5 times with washing solution (0.01M phosphate buffer with a pH of 7.2 + 0.05wt% Tween-20), and the amount of washing solution used for each wash is 250 μL / well. Finally, completely remove the liquid in the reaction well 20, control it on absorbent paper, and dry it at 37°C. Assemble and fix the upper sample loading structure body 100 of the qualified U-shaped reaction plate, put it into an aluminum foil bag, add a desiccant, vacuum seal it, and store it at 4°C for use.

[0098] (2) Detection

[0099] Take out the test kit body according to the detection amount and mark it. The remaining test kit body should be put back into the ziplock bag and sealed and stored at 2-8°C. Dilute the irregular antibody detection reagent (spectrum cells) with 0.9wt% sodium chloride solution to a 0.15% cell suspension for use. Add 150μL of exclusion chromatography to the corresponding sample chamber 111, centrifuge at 1000rpm for 10s, and centrifuge the exclusion chromatography into the reaction well 20. Add 50μL of diluent to the sample chamber 111, add 25μL of the plasma or serum sample to be tested, and then add 50μL of red blood cell suspension and mix well. Incubate at 37°C for 15min. Centrifuge at 1600rpm for 3min and 1400rpm for 5min. Determine the test results.

[0100] Result judgment criteria

[0101] The red blood cell immune complex spreads on the bottom surface of the U-shaped microplate under the action of a certain centrifugal force, forming a positive reaction; red blood cells cannot form immune complexes, and under the action of a certain centrifugal force, the red blood cells gather in the center of the bottom of the U-shaped microplate to form a cell "buckle", which is a negative reaction.

[0102] The above are only preferred embodiments of the present invention and are not intended to limit the present invention in any form. Any ordinary technician in the industry can smoothly implement the present invention as shown in the drawings and above. However, any equivalent changes, modifications and evolutions made by technicians familiar with the profession without departing from the scope of the technical solution of the present invention using the technical content disclosed above are all equivalent embodiments of the present invention. At the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still within the protection scope of the technical solution of the present invention.

Claims

1. A sample loading structure for an anti-human globulin test kit, comprising at least one sample loading tube (10); characterized in that: The sample adding tube (10) is a plastic part, comprising: A tube body (11); the tube body (11) is provided with a sample adding chamber (111) for adding a sample to be tested or a sample to be tested and a detection reagent for an anti-human globulin test; the bottom of the sample adding chamber (111) is provided with at least one through hole (112) for passing liquid under centrifugal action; A base (12) for supporting the tube body (11); The base (12) is provided with a fixing structure for fixing in the reaction hole (20) coated with anti-human globulin at the bottom of the reagent kit body (1).

2. The sample loading structure for the anti-human globulin test kit according to claim 1, characterized in that: A plurality of convex ribs (121) are evenly distributed on the outer peripheral side of the base, and are used to abut against the inner surface of the reaction hole (20) to achieve fixation.

3. The sample loading structure for the anti-human globulin test kit according to claim 1, characterized in that: The bottom surface of the sample adding chamber (111) is a concave curved surface.

4. The sample loading structure for the anti-human globulin test kit according to claim 1, characterized in that: The number of the through holes (112) is at least four; one of the through holes (112) is arranged at the center of the bottom of the sample loading chamber (111); and the remaining through holes (112) are distributed in a ring array around the center of the bottom of the sample loading chamber (111).

5. The sample loading structure for the anti-human globulin test kit according to claim 1, characterized in that: The number of the sample adding tubes (10) is eight or twelve in a row.

6. The sample loading structure for the anti-human globulin test kit according to claim 5, characterized in that: Two adjacent sample adding tubes (10) are provided with a first connecting piece (13).

7. The sample loading structure for the anti-human globulin test kit according to claim 6, characterized in that: The first connecting piece (13) is disposed laterally near the top of the sample adding tube (10) and is used for marking.

8. The sample loading structure for the anti-human globulin test kit according to claim 7, characterized in that: Two adjacent first connecting members (13) are arranged alternately to be close to two sides of the sample adding tube (10) respectively.

9. A kit, comprising a kit body (1), characterized in that: The kit body (1) comprises: The sample addition structure for an anti-human globulin test kit according to any one of claims 1 to 8; A U-shaped reaction plate (200) is provided with eight or twelve rows of reaction wells (20); the bottom of the reaction wells (20) is coated with anti-human globulin; and the reaction wells (20) are filled with a size exclusion chromatography separation medium; The sample adding structure body (100) is a structure of eight or twelve rows of sample adding tubes (10), which is fixed to the U-shaped reaction plate (200) via a base, and one of the sample adding tubes (10) is fixed to one of the reaction holes (20).

10. A detection method for anti-human globulin test, characterized in that: Using a kit as claimed in claim 9 to detect incomplete antibodies specifically comprises the following steps: S1, coating anti-human globulin at the bottom of the reaction well (20); S2, installing the sample adding tube (10) on the reaction hole (20); S3, adding a size exclusion chromatography separation medium to the sample tube (10), and centrifuging to allow the size exclusion chromatography separation medium to enter the reaction well (20); S4, adding the sample to be tested and the detection reagent to the sample tube (10), mixing or mixing and incubating; S5. Centrifuge and observe the results.

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