Method and reagent for specific identification of irregular blood group antibodies

Abstract

The present application relates to a novel method for specific identification of irregular blood type antibodies and a reagent thereof. The method comprises the following steps: (a) preparing a specific blood type antibody Fab fragment; (b) identifying the specificity of an incomplete antibody. Wherein, step (a) comprises taking a specific blood type antibody IgG, such as anti-D, anti-E, anti-C, anti-c, anti-e, anti-K, etc., treating it with papain or pepsin, and preparing the Fab fragment of the specific blood type antibody IgG. For the specific identification of a partial blood type system, papain or pepsin can also be used to prepare an IgM Fab fragment, or a mixture of IgG and IgM Fab fragments; step (b) comprises: (1) discovering irregular antibodies; (2) preliminarily inferring the specificity of irregular antibodies based on the serological behavior characteristics of the antibodies being tested; (3) preparing corresponding sensitized red blood cells; (4) reacting the tested serum with sensitized red blood cells or corresponding antigen-positive red blood cells; (5) determining the result. The reagent is the specific blood type antibody Fab fragment prepared in step (a) of the method for specific identification of irregular antibodies.

Description

Technical Field

[0001] The invention belongs to the technical field of irregular blood type antibody detection and identification, and in particular relates to a novel irregular blood type antibody specific identification method and an irregular blood type antibody specific identification reagent. Background Art

[0002] Irregular antibodies in serum are also called unexpected antibodies. They refer to blood type antibodies that do not conform to the Landsteiner rule of the ABO blood type system, that is, blood type antibodies other than anti-A, anti-B and anti-AB. There are two types of normal human blood type antibodies: IgG and IgM. According to current clinical research, most of the ABO blood type system anti-A and anti-B antibodies are IgM type, and irregular antibodies are mostly IgG type. Anti-A and anti-B IgM antibodies can specifically bind to the A and B antigens on the surface of red blood cells in saline media and produce visible agglutination. This method is traditionally used clinically to identify human ABO blood types. However, traditional saline blood type and cross-matching tests are only effective for the ABO blood type system, and are ineffective for many other blood type systems. In cases where blood transfusion is required or pregnancy occurs, it is necessary to detect irregular antibodies in the blood to prevent irregular antibodies from binding to corresponding antigens during transfusion or production to produce immune reactions. Otherwise, it may cause fever and chills at the mildest, or hemolysis at the worst, which is life-threatening. Irregular antibodies can cause hemolytic disease of the newborn, affecting the development of the newborn's organs and intelligence, and in severe cases can threaten the newborn's life. Therefore, blood transfusion recipients and pregnant women need to be screened for irregular antibodies, and blood donors should also be screened to prevent transfusing blood containing irregular antibodies to others.

[0003] At present, the screening and identification of irregular antibodies uses a group of O-type red blood cells with known antigenic phenotypes, and uses different reaction media (saline, anti-human globulin medium, enzyme medium, polybrene, etc.) at different temperatures to screen whether the tested serum contains irregular antibodies. If so, the specificity of the irregular antibodies should be further identified. If this antibody has clinical significance, red blood cells without corresponding antigens should be used for transfusion to avoid transfusion reactions and ensure transfusion safety. If the tested serum does not react with its own cells or screening cells, it means that there are no irregular antibodies in the serum. If the tested serum does not agglutinate with its own cells, but agglutinates with one or several screening cells, it means that it contains irregular antibodies. If the antibody screening is positive, it is necessary to further identify the specificity of irregular antibodies. The serum of those who are positive for irregular antibodies to red blood cells is further reacted with standard spectrum cells, and the antibody specificity is identified based on the reaction pattern. When the tested serum reacts negatively with all the spectrum cells, the antibody screening test is repeated. If it reacts positively with a certain screening cell, the antibody may be an antibody to a low-frequency antigen or a private antigen; when the tested serum reacts positively with all the spectrum cells, the antibody may be directed against a high-frequency antigen or a public antigen, or there may be mixed antibodies with multiple specificities, which require multiple methods of treatment. When the tested serum reacts positively with some spectrum cells and negatively with other spectrum cells, it is necessary to use methods such as negative exclusion, positive comparison, antigen evidence, and probability calculation to make a judgment (Experimental Technology of Transfusion Immunology and Hematology, edited by Liu Jinghan and Lan Jiongcai, People's Medical Publishing House, 2011, pages 57-62).

[0004] Combining the tested serum and spectrum cell reaction pattern in Table 1, the antibody specificity determination rules are as follows:

[0005] 1. The serum does not react with cells No. 7 and No. 9, and the antibodies corresponding to the antigens contained in these two cells are excluded;

[0006] 2. The serum reacts positively with cells 1, 2, 3, 4, 5, 6, 8, and 10, and the antibodies corresponding to the antigens not contained in these eight cells are also excluded;

[0007] 3. According to the above exclusion results, the most likely possibility is anti-D or anti-E of the Rh blood group system. Use anti-D and anti-E to detect D and E antigens on the patient's cells. If the D antigen is positive, it cannot produce anti-D antibodies; similarly, if the E antigen is positive, anti-E can be ruled out;

[0008] 4. If both D and E antigens are negative and the specificity cannot be determined, it is necessary to further select two O-type red blood cells with ccdEe phenotype for supplementary testing, or select another set of spectrum cells with multiple ccdEe cells to repeat the test;

[0009] 5. Probability calculation: In order to exclude the contingency of the identification results, it is necessary to perform statistical processing based on the number of positive and negative reactions between the tested serum and the spectrum cells. Only when P≤0.05 can a clear result be obtained; when P>0.05, it is an ambiguous result and the number of spectrum cells needs to be increased to continue the test. The Fisher method calculates the probability through a 2×2 four-grid table. The positive and negative reactions of the tested serum are related to the presence or absence of specific antigens on the spectrum cells. P=(a+b)! ×(c+d)! ×(a+c)! ×(b+d)! / n! ×a! ×b! ×c! ×d!

[0010] Table 1 Examples of the reaction patterns of the tested serum and spectrum cells

[0011]

[0012] Note: 10 blood type systems and 25 antigens (antibodies) are quoted from "Experimental Techniques of Transfusion Immunology and Hematology", edited by Liu Jinghan and Lan Jiongcai, People's Medical Publishing House, 2011, pages 57-62.

[0013] In summary, the existing methods for identifying the specificity of irregular antibodies mainly use standard spectrum cells, and the establishment and preservation of standard spectrum cells are very cumbersome and difficult, and have many limitations:

[0014] 1. Difficulty in establishment: The standard spectrum cells are composed of 8 to 16 O-type red blood cells. These cells must contain as many antigens as possible, at least more than 20 major antigens of the Rh, MNSs, Kell, Diego, Kidd, P, Duffy and Lewis blood group systems. At the same time, it must contain red blood cells that lack certain antigens, such as Rh-negative O-type red blood cells (panda blood). This blood type is less than 1‰ among Han blood donors, and it is very difficult to get these rare blood type people to donate blood regularly and keep them healthy.

[0015] 2. Difficulty in storage and transportation: After carefully selected O-type spectrum cell donors donate blood, if it is to be stored for a long time, the temperature must be below -80℃. This temperature is difficult to maintain during transportation, especially since many spectrum cells are now from abroad. It is even more difficult to maintain a temperature below -80℃ during long-distance transportation. Domestic transportation is transported and stored at 2℃ to 8℃. Although the temperature is easy to control, spectrum cells can only be stored for about 3 months at this temperature. When they reach the hands of customers, the storage period is often less than 3 months.

[0016] 3. The test results are unstable: First, the serological behavior of some antibodies has a dose effect, that is, the reaction with homozygous cells is relatively strong, and the reaction with heterozygous cells is relatively weak or no reaction, which can easily miss some weak antibodies with clinical significance; when the spectrum cells are stored at 2℃~8℃, the closer to the end of the storage period, the weaker the antigenicity of the cells, and some weak antibodies or antibodies with a dose effect may affect the detection rate.

[0017] 4. Since this method uses cells with known antigen spectrum to react with the serum under test containing unexpected antibodies, it mainly uses the method of elimination and inference to identify the specificity of the antibody, and the so-called "known antigen" is actually impossible to do (there are more than 30 blood type systems known for red blood cells, including hundreds of antigens). The final result is only a statistically significant result (the smaller the P value, the more reliable the identification result).

[0018] The method of using the standard spectrum cell method to detect irregular antibodies is relatively cumbersome and the detection cost is also high. At present, there are some methods that have improved the detection of irregular antibodies, but they are still not perfect. In order to improve the irregular antibody detection method, this application introduces the classic technology in traditional immunology into the experiment of blood type serology, modifies and processes the blood type antibody IgG molecules, and establishes a new blood type irregular antibody specific identification method and reagents. Summary of the invention

[0019] The present application relates to a method for specific identification of irregular blood type antibodies, which comprises the following steps:

[0020] (a) preparing specific blood type antibody Fab fragments;

[0021] (b) Characterization of the specificity of incomplete antibodies.

[0022] Wherein, step (a) further comprises taking specific blood type antibody IgG, such as anti-D, anti-E, anti-c, anti-e, anti-K, treating it with papain or pepsin to prepare the specific blood type antibody Fab fragment. For the specific identification of some blood type systems, papain or pepsin can also be used to prepare IgM Fab fragments, or to prepare a mixture of IgG and IgM Fab fragments for standby use. The specific blood type antibody IgG is selected from self-prepared polyclonal antibodies or monoclonal antibodies, or commercially available blood type reagents.

[0023] Wherein, step (b) further comprises:

[0024] (1) Irregular antibodies were discovered;

[0025] (2) Preliminary inference of the specificity of irregular antibodies;

[0026] (3) preparing sensitized red blood cells;

[0027] (4) reacting the tested serum with sensitized red blood cells or red blood cells positive for the corresponding antigen;

[0028] (5) Determination result.

[0029] Wherein, sub-step (1) in step (b) includes finding irregular antibodies in the blood during cross-matching, antibody screening, neonatal hemolytic disease testing and transfusion reaction testing.

[0030] Wherein, sub-step (2) in step (b) includes preliminarily estimating the range of antibody specificity based on the blood type serological characteristics of the antibody; it further includes estimating the specificity of the irregular antibody by examining the relevant antigens of the tested red blood cells.

[0031] Wherein, sub-step (3) in step (b) includes incubating the Fab of the corresponding antibody with the known corresponding antigen-positive red blood cells under the conditions of specific temperature, ionic strength and pH value to fully sensitize them; then washing with physiological saline for 3 times, preparing a 2% to 5% cell suspension for standby use. For example: for the ccdee phenotype of the red blood cells to be tested, select anti-D (Fab) and D antigen-positive red blood cells for incubation, anti-E (Fab) and E antigen-positive red blood cells for incubation, or anti-C (Fab) and C antigen-positive red blood cells for incubation to prepare the sensitized red blood cells.

[0032] Among them, sub-step (4) in step (b) includes taking 2 drops of the test serum and one drop of the corresponding 2% to 5% suspension of sensitized red blood cells, adding them into test tubes respectively, and reacting them in saline, enzyme medium, polybrene medium or anti-human globulin medium at 2°C to 39°C, and at the same time performing parallel control experiments with the test serum and corresponding antigen-positive red blood cells.

[0033] Wherein, sub-step (5) in step (b) includes: ① When the test serum agglutinates with the control cells but not with the sensitized red blood cells, it indicates that the corresponding antigen on the red blood cells is blocked by Fab, and the specificity of the antibody in the test serum is consistent with the specificity of Fab; ② When the test serum agglutinates with the control experiment but also with the sensitized red blood cells, there are two possibilities: one is that the antibody specificity of the test serum is inconsistent with the specificity of the Fab segment; the other is that the test serum contains more than two complex antibodies, and further identification is required, that is, sensitizing the red blood cells with different specific Fab combinations, and then observing and analyzing the results to make a judgment; ③ When the control experiment does not agglutinate, it indicates that there is an error in the experimental process and the experimental results are unreliable. It is necessary to recheck the experimental conditions or replace appropriate control red blood cells and repeat the experiment.

[0034] The present application has at least the following beneficial effects: the present application ingeniously introduces the classic technology in traditional immunology into the experiment of blood type serology, modifies and processes the blood type antibody IgG or IgM molecules, creates a new method for simple, convenient and accurate blood type antibody specific identification, and develops a new reagent (antibody Fab fragment) for blood type identification; enables the specific identification of antibodies in immunohematology experiments to transition from the identification of unknown antibodies using known antigens (corresponding spectrum cells) to the identification of unknown antibodies using known antibodies; avoids the limitations of a series of processes such as spectrum cell establishment, preservation, transportation and result judgment. Moreover, the identification method of the present application can completely determine the specificity of irregular antibodies, which is significantly superior to the existing standard spectrum cell method that determines the specificity of irregular antibodies by estimating statistical probability (P≦0.05). In addition, the reagent (antibody Fab fragment) of the new method belongs to the serum antibody class, and the storage time at 2°C to 8°C can reach more than two years, which is longer than the storage time of standard spectrum cell reagents, bringing convenience to experimental operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The present application is described in detail below in conjunction with the accompanying drawings, but the accompanying drawings are only used to describe and explain the present application and do not limit the scope of protection of the present application.

[0036] Figure 1 A schematic diagram illustrating an anti-human globulin experimental method for detecting incomplete antibodies in the prior art;

[0037] Figure 2 A schematic diagram illustrating the heavy chain and light chain composition of an antibody;

[0038] Figure 3 A schematic diagram illustrating the domain structure of antibody heavy and light chains;

[0039] Figure 4 Schematic diagram illustrating papain hydrolysis of antibodies;

[0040] Figure 5 Schematic diagram illustrating pepsin hydrolysis of an antibody;

[0041] Figure 6 Schematic diagram illustrating the principle of the method of the present application. RBC represents red blood cells with D antigen. The white Y-shaped structure in the figure is anti-D antibody, such as Figure 6 Indicated in A. The gray Y-shaped substance with a similar structure to the anti-D antibody is anti-human globulin, such as Figure 6 A and 6B are indicated;

[0042] Figure 7 Antigen pattern table of antibody screening red blood cell kit (production batch number: 20197056);

[0043] Figure 8 Antigen pattern table for antibody screening red blood cell kit (production batch number: 20207045). DETAILED DESCRIPTION

[0044] In order to make the purpose, technical solution and advantages of the present application clearer, the technical solution of the present application will be clearly and completely described below. Based on the specific implementation methods in the present application, all other implementation methods obtained by those skilled in the art without creative work belong to the scope of protection of the present invention. The above content and other aspects of the present invention will be further described below, where: unless otherwise specified or defined, all terms used have the general meaning in the art and are well known to those skilled in the art (for example, see "Transfusion Testing", edited by Xu Qunfang and He Zhi, China Medical Press, 2019).

[0045] In the present application, the term "antibody" refers to an important effector molecule that mediates humoral immunity. It is a glycoprotein produced by B cells that proliferate and differentiate into plasma cells after receiving antigen stimulation, and is mainly present in body fluids such as serum.

[0046] In the present application, the term "blood type antibody" refers to the immunoglobulins produced by the body after being stimulated by blood type antigens, when B cells are activated, proliferate and differentiate into plasma cells, which can bind to the corresponding antigens and induce an immune response.

[0047] In this application, the term "regular antibody" refers to the presence of a certain antigen on the surface of red blood cells, and antibodies that are not directed against the antigen appear regularly in the blood, which are called regular antibodies. Among all blood group systems, only the production of ABO blood group antibodies is regular.

[0048] In the present application, "irregular antibodies" refer to irregular antibodies produced by blood group systems other than the ABO blood group system, or unexpected antibodies.

[0049] In the present application, "complete antibody" is also called saline antibody, which refers to an antibody that can directly agglutinate red blood cells in a saline medium, and most of them are IgM antibodies.

[0050] In the present application, "incomplete antibodies" refer to antibodies that do not show visible agglutination reaction in saline medium after binding to antigens (red blood cells), but only show agglutination reaction in special media, most of which are IgG antibodies.

[0051] In the present application, "natural antibodies" refer to blood type antibodies that exist in the blood without clear antibody stimulation (for example, blood transfusion, pregnancy, etc.), such as antibodies of the ABO system. Natural antibodies are mostly IgM antibodies and complete antibodies.

[0052] In this application, "immune antibodies" refer to blood type antibodies produced after relatively clear antigen stimulation, such as: blood transfusion, pregnancy, injection, etc. Such antibodies are mostly IgG class and incomplete antibodies.

[0053] In the present application, the "Fab fragment" and "Fc fragment" of an antibody refer to two completely identical antigen-binding fragments (Fab) and one crystallizable fragment (Fc) produced after papain hydrolyzes the antibody molecule.

[0054] In this application, "spectrum cells" or "matched cells" refer to a group of reagent red blood cells used for specific identification of blood group antibodies. A set of spectrum cells should include as many antigens as possible, as well as some red blood cells lacking certain antigens. Generally, it consists of 8 to 16 types of red blood cells, which can identify antibodies of Rh, MNSs, Kell, Diego, Kidd, P, Duffy and Lewis blood group systems.

[0055] Introduction to the principles of existing methods for detecting incomplete antibodies

[0056] Under normal circumstances, there is sialic acid on the surface of the red blood cell membrane, which carries a negative charge, causing the red blood cells to repel each other and keep adjacent red blood cells at a certain distance; the span of incomplete antibody (IgG) molecules is too small to directly connect to adjacent red blood cells, causing agglutination visible to the naked eye.

[0057] Principle of enzyme experiment: Enzymes can destroy sialic acid on the surface of red blood cells, reduce the negative charge they carry, shorten the distance between adjacent red blood cells, and allow incomplete antibodies (IgG) to directly connect to adjacent red blood cells and cause agglutination;

[0058] Principle of polybrene medium test: Polybrene is a high-valent cationic quaternary ammonium salt with multiple cation groups in the solution, generating multiple positive charges, which can neutralize the negative charge on the surface of red blood cells, shorten the distance between adjacent red blood cells, and enable incomplete antibodies (IgG) to directly connect to adjacent red blood cells and cause agglutination;

[0059] Principle of anti-human globulin test: The anti-human globulin test is a classic method invented by Coombs et al. in 1945. It is mainly used to detect incomplete antibodies (IgG) in blood immunology. IgG is a 7S monomer structure with a small molecular weight and a short molecular span. It can only sensitize red blood cells in saline medium and cannot produce visible agglutination reactions. After adding anti-human globulin reagent, the latter's Fab fragment binds to the Fc fragment of IgG coated on the surface of red blood cells, thereby producing red blood cell agglutination through the bridging effect of anti-human globulin molecules (such as Figure 1As shown in (see "Clinical Transfusion Laboratory Technology", edited by Hu Lihua et al., People's Medical Publishing House, March 2015, pages 34-36).

[0060] The method principle of this application

[0061] Antibodies are important effector molecules that mediate humoral immunity. They are glycoproteins produced by B cells that differentiate into plasma cells after receiving antigen stimulation. Immunoglobulins refer to globulins with antibody activity or chemical structure. The basic structure of immunoglobulins is a symmetrical structure composed of four peptide chains in a Y shape, consisting of two heavy chains (H chains) and two light chains (L chains) connected by disulfide bonds between the chains (such as Figure 2 As shown in Figure 1.1.1. Each heavy and light chain has one end called the amino terminus (N terminus) and the other end called the carboxyl terminus (C terminus). At the N terminus, 1 / 2 of the light chain and 1 / 4 of the heavy chain are called the variable region (V region). The order of amino acids in the V region varies with the specificity of the antibody, recognizing and binding to specific antigens. The rest of the light and heavy chains are called the stable region (C region) (as shown in Figure 1. ... Figure 3 shown).

[0062] Different fragments can be obtained by using different proteolytic enzymes to hydrolyze immunoglobulin molecules. For example, using papain to hydrolyze IgG can produce three fragments, namely two antigen-binding fragments Fab fragments and one crystallizable fragment Fc fragment. A Fab fragment is monovalent and can bind to the antigen but does not produce agglutination or precipitation reactions. The Fc fragment has no antigen-binding activity (such as Figure 4 Pepsin acts on the C-terminus of the disulfide bond and hydrolyzes it to obtain a F(ab′)2 fragment and some small fragments pFc′, such as Figure 5 As shown (see Molecular Biology, edited by Chen Qimin, Wang Jinzhong, and Geng Yunqi, Nankai University Press, December 2004, pp. 371-374; Experimental Techniques of Transfusion Immunology and Hematology, edited by Liu Jinghan and Lan Jiongcai, People's Medical Publishing House, 2011, pp. 5-6).

[0063] The specificity of the blood type antibody IgG or IgM is known. It is first treated with an enzyme such as papain to hydrolyze it into Fab and Fc segments, such as Figure 4As shown. Then, the red blood cells positive for the corresponding antigen are incubated with the Fab segment of the antibody. Since the Fab segment still has the activity of the antibody, it binds to the antigen and produces red blood cells sensitized by the Fab segment. When such sensitized red blood cells are reacted with the test serum in saline, enzyme medium, polybrene medium or anti-human globulin medium, and the same cells without Fab segment sensitization are used as a control, if the test serum is consistent with the specificity of the known blood type antibody, since the corresponding antigen has been blocked by Fab binding, no agglutination reaction will occur, while the control cells will still show visible agglutination reaction. Therefore, it is concluded that the test serum contains antibodies with the same specificity as the Fab segment. Taking red blood cells RBC containing D antigen as an example, when the D antigen red blood cells bind to the anti-D antibodies in the test serum, the cells agglutinate under the bridging effect of the anti-human globulin molecules, as shown in FIG. Figure 6 As shown in A Figure 6 A shows a control experiment, taking anti-D as an example); after the D antigen red blood cells bind to the anti-D (Fab) fragment, since the D antigen has been blocked, the sensitized red blood cells RBC cannot bind to the anti-D antibody or the anti-human globulin molecule, and thus cannot produce agglutination, such as Figure 6 As shown in B.

[0064] Specific steps of this application method

[0065] 1. Preparation of specific antibody Fab fragments:

[0066] Take specific blood type antibody IgG (you can prepare polyclonal or monoclonal antibodies yourself or buy commercial blood type reagents), such as anti-D, anti-E, anti-c, anti-e, anti-K, etc. For the specific identification of some blood type systems, you can also use papain or pepsin to prepare IgM Fab fragments, or prepare a mixture of IgG and IgM Fab fragments. Treat with papain or pepsin to prepare Fab of specific blood type antibodies, such as anti-D (Fab), for use. The method refers to "Antibody Preparation and Use Laboratory Guide", 2nd edition, edited by GC Howard and MR Kaiser, Science Press, pages 208-213.

[0067] 2. Specific process of incomplete antibody specificity identification:

[0068] (1) Discovery of unexpected antibodies: If irregular blood type antibodies are found in the blood during cross-matching, antibody screening, neonatal hemolytic disease testing and transfusion reaction testing, further identification is required.

[0069] (2) Based on the blood type serological characteristics of the antibody, the range of antibody specificity can be preliminarily estimated, for example, 2°C to 39°C, such as 4°C, 22°C, and 37°C. The reaction characteristics in saline, enzyme media, polybrene media, anti-human globulin and other media can be preliminarily estimated to be the approximate range of blood type antibody specificity.

[0070] (3) Check the relevant antigens of the tested red blood cells to further infer the specificity of the unexpected antibody. If such antigens are present on the tested red blood cells, the corresponding antibodies should not be produced in the serum. For example, if the latter red blood cells are ccdee, it is highly suspected that the antibodies are anti-D, or anti-E and anti-C, but not anti-C or anti-E.

[0071] (4) Use the Fab of the most likely corresponding antibody to incubate with the known corresponding antigen-positive red blood cells under specific temperature, ionic strength, and pH conditions to fully sensitize them. Then wash with physiological saline three times and prepare a 2% to 5% cell suspension for later use. For example: if the red blood cells of the subject are of ccdee phenotype, select anti-D (Fab) to incubate with D-positive red blood cells, anti-E (Fab) to incubate with E antigen-positive red blood cells, anti-C (Fab) to incubate with C antigen-positive red blood cells, and so on.

[0072] (5) Take 2 drops of the test serum and one drop of the corresponding 2% to 5% suspension of sensitized red blood cells, add them into test tubes respectively, and react them at 2°C to 39°C (for example, at 4°C, 22°C, 37°C) in a medium such as saline, enzyme medium, polybrene medium, and anti-human globulin. At the same time, use the test serum and red blood cells positive for the corresponding antigen for parallel control experiments (see "Clinical Transfusion Laboratory Technology", edited by Hu Lihua, People's Medical Publishing House, March 2019, pages 33-39).

[0073] (6) Result determination:

[0074] ① The tested serum agglutinated with the control cells but not with the sensitized cells, indicating that the corresponding antigens on the red blood cells were blocked by Fab and the specificity of the antibodies in the tested serum was consistent with that of Fab.

[0075] ② Agglutination occurred in the control experiment, but agglutination also occurred with sensitized cells. There are two possibilities: one is that the specificity of the serum antibody being tested is inconsistent with the specificity of the Fab segment; the other is that the serum contains more than two complex antibodies, which requires further identification. Use Fab combinations of different specificities to sensitize the red blood cells separately, and then observe and analyze the results to make a judgment.

[0076] ③If no agglutination occurs in the control experiment, it indicates that something went wrong in the experimental process and the experimental results are unreliable. It is necessary to recheck the experimental conditions or replace appropriate control red blood cells and repeat the experiment.

[0077] Reagents for this application

[0078] The reagent of the present application is the Fab fragment of the specific blood type antibody prepared by treating the specific blood type antibody with papain or pepsin in step 1 of the method of the present application.

[0079] The above is a detailed introduction to the specific implementation methods of the present invention. For those skilled in the art, several improvements and modifications may be made to the present invention without departing from the principles of the present invention. These improvements and modifications also fall within the scope of protection of the claims of the present invention.

[0080] Example

[0081] Example 1. A case of anti-D found in blood samples sent for clinical examination

[0082] On January 17, 2020, a cross-matching case was received from Shengjing Hospital Affiliated to China Medical University (Huaxiang). The patient was a 76-year-old male. During the blood matching process, it was found that the patient's antibody screening was positive and was identified as anti-D. The specific identification process of the irregular antibody is as follows:

[0083] 1. Sample reception and preprocessing

[0084] (1) The experimenter reviews and checks whether the blood sample of the recipient meets the requirements.

[0085] (2) Check whether the blood sample label is consistent with the test submission form.

[0086] (3) After the experimenter confirms that the blood sample and the test form meet the requirements, he / she shall fill in the "Record of Receipt, Storage and Destruction of Specimens of the Institute of Transfusion Medicine (Blood Matching)".

[0087] (4) Fill in the "Difficult Cross-matching Experiment Record" based on the blood transfusion application form.

[0088] (5) Treatment of blood samples from recipients: After adding the coagulant, the blood sample is centrifuged and separated, and the upper serum can be used directly for the experiment. Centrifuge and separate the anticoagulated blood, take 2-3 drops of anticoagulated red blood cells, wash them with saline for 3 times, and prepare a 2%-5% red blood cell suspension (1 volume of packed red blood cells plus 16-40 volumes of saline) for later use.

[0089] 2. Identification of the recipient's ABO and Rh blood types

[0090] (1) ABO blood typing (test tube method)

[0091] Positive typing: Take two clean test tubes, mark them with anti-A and anti-B respectively, use a dropper to add 1 drop of anti-A and anti-B typing serum to the bottom of the test tube, then use a dropper to add 1 drop of 2% to 5% red blood cell suspension of the recipient, mix well. Centrifuge at 900 to 1000 g for 15 seconds and observe the results.

[0092] Reverse typing: Take another 4 clean test tubes and label them as A cells, B cells, O cells and own cells. Use a dropper to add 2 drops of the patient's plasma to each of the above labeled test tubes. Then use a dropper to add 1 drop of A, B, O reverse typing red blood cell suspension and 2% to 5% red blood cell suspension of the patient, respectively, and mix well. Centrifuge at 900-1000g for 15 seconds and observe the results.

[0093] (2) Rh(D) blood typing (test tube method)

[0094] Take three other test tubes, label the recipient, Rh(D) negative and positive controls, and add 1 drop of IgM+IgG anti-Rh(D) serum to each tube. Add 1 drop of 2%-5% recipient red blood cell suspension, Rh(D) negative and positive standard red blood cell suspension to each test tube, and mix gently. After leaving at room temperature for 1 minute, centrifuge at 900-1000g for 15 seconds to observe the results.

[0095] (3) The results of ABO and Rh(D) blood type identification of the recipient are shown in Table 2:

[0096] Table 2. Results of ABO and Rh(D) blood typing by in vitro method

[0097]

[0098] The results showed that the blood recipient was type A blood and Rh(D) negative.

[0099] 3. Blood recipient serum antibody screening (gel card method)

[0100] (1) Prepare I, II, and III standard antibody screening cells (0.8%), equilibrate diluent No. 2 at room temperature, prepare samples and label Diameka.

[0101] (2) Add 50 μl of antibody screening I, II, and III cells to each of the three tubes, and add 25 μl of the subject's plasma. Incubate at 37°C for 15 minutes.

[0102] (3) Centrifuge for 10 minutes using the matching centrifuge and read and record the results.

[0103] The experimental results are shown in Table 3:

[0104] Table 3. Antibody screening (gel card method) results

[0105]

[0106] 4. Direct anti-human globulin test in blood recipients

[0107] (1) Prepare EDTA-anticoagulated blood sample with normal saline to a concentration of 2% to 5% red blood cells.

[0108] (2) Add 1 drop of 2% to 5% red blood cell suspension to each of the test tube and the control tube.

[0109] (3) Wash with saline 3 to 4 times. Completely remove the supernatant during the last wash.

[0110] (4) Immediately add 1 drop of anti-human globulin reagent to the test tube and 1 drop of saline or 6% albumin to the control tube, mix well. Centrifuge at 900-1000g for 15 seconds. Observe the agglutination, score and record the results.

[0111] (5) If no agglutination is observed in the assay tube, add IgG-sensitized red blood cells to the test tube containing the antiglobulin reagent and centrifuge at 900-1000 g for 15 seconds. Observe and record the results to confirm the validity of the negative result.

[0112] The direct antibody result is negative. The antibody screening result is positive, such as Figure 7 As shown in the attached antibody screening red blood cell kit antigen pattern table, combined with the positive and negative typing experiments, the tested serum reacted negatively with type O red blood cells in room temperature saline, and the tested cells reacted negatively with anti-D antibodies, revealing that there is a high possibility of incomplete anti-D antibodies in the recipient's plasma. Anti-D (Fab) inhibition experiments are required for further confirmation.

[0113] 5. Antibody specificity identification (anti-D (Fab) agglutination inhibition test)

[0114] (1) Take one drop of O-type red blood cells known to be Rh(D) positive, wash them three times with 0.9% saline, and prepare a 2% to 5% red blood cell suspension.

[0115] (2) Take two small test tubes, one labeled as Fab and the other labeled as control. Add 1 drop of the above 2% to 5% red blood cell suspension to each of the two test tubes, 2 drops of anti-D (Fab) to the Fab tube, and 2 drops of 0.9% saline to the control tube. Mix well and place in a 37°C water bath for 30 minutes.

[0116] (3) Wash the cell suspensions in the two test tubes three times with 0.9% saline and finally add 1 drop of saline to each tube to make a 2% to 5% suspension.

[0117] (4) Add 2 drops of serum to be tested to each of the two test tubes, mix thoroughly, and place in a 37°C water bath for 30 minutes.

[0118] (5) Wash with 0.9% saline for 3 times, drain the water for the last time, add 2 drops of anti-human globulin serum, mix thoroughly, centrifuge at 900-1000g for 15 seconds, observe whether hemolysis occurs, gently resuspend the cells, observe the agglutination reaction, and record the room temperature reaction results. The experimental results are shown in Table 4:

[0119] Table 4. Anti-D (Fab) agglutination inhibition test results

[0120]

[0121] The above experimental results show that the antibodies in the tested serum are inhibited by anti-D (Fab), indicating that the antibody specificity is consistent with anti-D, that is, the antibodies in the tested serum are anti-D.

[0122] 6. Verification of antibody specificity identification results

[0123] In order to further prove the accuracy of antibody specificity identification (anti-D (Fab) inhibition experiment), combined with the actual situation of the sample and laboratory, it was decided to use the spectrum cells produced by Shanghai Blood Biotechnology Co., Ltd. with batch number 20191205 for further antibody identification. The identification method used the standard spectrum cell microcolumn gel method with higher sensitivity.

[0124] (1) Label Diameca and dilute the commercially available spectrum cells numbered 1-10 with diluent No. 2 that has been equilibrated at room temperature to a final concentration of 0.8%.

[0125] (2) Add 50 μl of the diluted spectrum cells numbered 1-10 to the marked diamica columns, and then add 25 μl of the subject's plasma.

[0126] (3) Incubate at 37°C for 15 minutes.

[0127] (4) Centrifuge for 10 minutes using a matching centrifuge, and record and interpret the analysis results. The experimental results are shown in Table 5: After antibody identification, it is suspected to be anti-D.

[0128]

[0129] 7. Further identification of Rh phenotype

[0130] Take 5 small test tubes, mark them with anti-D, anti-C, anti-c, anti-E, and anti-e, add 1 drop of anti-D, anti-C, anti-c, anti-E, and anti-e serum, and then add 1 drop of 2% to 5% red blood cell suspension of the subject, mix gently. Leave at room temperature for 1 minute, centrifuge at 900 to 1000g for 15 seconds, observe and record the results.

[0131] The results of Rh phenotype identification are shown in Table 6:

[0132] Table 6. Rh phenotype identification results

[0133]

[0134] The experimental results showed that the subject's red blood cells agglutinated with anti-C, anti-c, anti-E and anti-e sera, and his Rh phenotype could be determined to be CcdEe.

[0135] The results of the internal quality control and external quality assessment of this laboratory showed that no out-of-control problems occurred, and the negative and positive control results of each part of the experiment were normal. After comparative analysis, the subject had the same antibody anti-D, which did not completely rule out the presence of other antibodies in the plasma. The blood matching method and steps were strictly carried out in accordance with the standard operating procedures of the Institute of Transfusion Medicine, and finally the blood of the donor with the same phenotype as the patient was selected for blood distribution.

[0136] Example 2. A case of anti-E found in a blood sample submitted for clinical examination

[0137] On December 5, 2020, a cross-matching case was received from the Affiliated Hospital of Liaoning University of Traditional Chinese Medicine. The patient was a 74-year-old woman. During the blood matching process, it was found that the patient's antibody screening was positive and was suspected to be anti-E. The identification process is as follows:

[0138] 1. Sample reception and preprocessing

[0139] Same as Example 1.

[0140] 2. Identification of the recipient's ABO and Rh blood types

[0141] (1) ABO blood typing (test tube method)

[0142] The methods of positive setting and negative setting are the same as those in Example 1.

[0143] (2) Rh blood typing (test tube method)

[0144] Take another 7 test tubes, mark the recipient, Rh(D) negative and positive controls, and add 1 drop of IgM+IgG anti-Rh(D), anti-E, anti-C, anti-e, and anti-c serum to each tube. Add 1 drop of 2% to 5% recipient red blood cell suspension, Rh(D) negative and positive standard red blood cell suspension to each test tube, and mix gently. After standing at room temperature for 1 minute, centrifuge at 900-1000g for 15 seconds to observe the results.

[0145] The results of ABO and Rh(D) blood type identification of the recipient are shown in Table 7:

[0146] Table 7. ABO and Rh(D) blood type identification results of the recipient

[0147]

[0148] The results showed that the blood recipient had type A blood and Rh phenotype was CcDee.

[0149] 3. Blood recipient serum antibody screening (gel card method)

[0150] (1) Prepare I, II, and III standard antibody screening cells (0.8%), equilibrate diluent No. 2 at room temperature, prepare samples and label Diameka.

[0151] (2) Add 50 μl of antibody screening I, II, and III cells to each of the three tubes, and add 25 μl of the subject's plasma. Incubate at 37°C for 15 minutes.

[0152] (3) Centrifuge for 10 minutes using the matching centrifuge and read and record the results.

[0153] The experimental results are shown in Table 8.

[0154] Table 8. Results of serum antibody screening (gel card method) of blood recipients

[0155]

[0156] 4. Conduct a direct anti-human globulin test on the recipient

[0157] (1) Prepare EDTA-anticoagulated blood sample with normal saline to a concentration of 2% to 5% red blood cells.

[0158] (2) Add 1 drop of 2% to 5% red blood cell suspension to each of the test tube and the control tube.

[0159] (3) Wash with saline 3 to 4 times. Completely remove the supernatant during the last wash.

[0160] (4) Immediately add 1 drop of anti-human globulin reagent to the test tube and 1 drop of saline or 6% albumin to the control tube, mix well. Centrifuge at 900-1000g for 15 seconds. Observe the agglutination, score and record the results.

[0161] (5) If no agglutination is observed in the assay tube, add IgG-sensitized red blood cells to the test tube containing the antiglobulin reagent and centrifuge at 900-1000 g for 15 seconds. Observe and record the results to confirm the validity of the negative result.

[0162] The experimental results are as follows Figure 8 The antigen pattern of the antibody screening red blood cell kit is shown in the table.

[0163] The direct antibody result was negative, and the 37℃ saline was also negative. The I and III cells in the anti-human globulin medium showed positive reactions, and the II cells showed negative reactions, revealing that the tested serum contained specific isotype incomplete antibodies. According to the serological behavior of the tested serum antibodies, and the patient's red blood cell Rh phenotype was CcDee, it is very likely that the antibody is anti-E, and further confirmation is required.

[0164] 5. Antibody specificity identification (anti-E (Fab) agglutination inhibition test)

[0165] (1) Take one drop of O-type red blood cells with a known phenotype of CcDEE (these cells can be screened by yourself using the corresponding standard serum or selected from commercially available standard spectrum cells, but the most common method is to screen them by yourself according to your needs. In this experiment, you can choose O-type red blood cells that are positive for E antigen, and you don’t need to pay attention to other antigens). Wash it three times with 0.9% saline and prepare a 2% to 5% red blood cell suspension.

[0166] (2) Take two small test tubes, one labeled as Fab and the other labeled as control. Add 1 drop of the above 2% to 5% red blood cell suspension to each of the two test tubes, 2 drops of anti-E (Fab) to the Fab tube, and 2 drops of 0.9% saline to the control tube. Mix well and place in a 37°C water bath for 30 minutes.

[0167] (3) Wash the cell suspensions in the two test tubes three times with 0.9% saline and finally add 1 drop of saline to each tube to make a 2% to 5% suspension.

[0168] (4) Add 2 drops of serum to be tested to each of the two test tubes, mix thoroughly, and place in a 37°C water bath for 30 minutes.

[0169] (5) Wash with 0.9% saline for 3 times, drain the water for the last time, add 2 drops of anti-human globulin serum, mix thoroughly, centrifuge at 900-1000g for 15 seconds, observe whether hemolysis occurs, gently resuspend the cells, observe the agglutination reaction, and record the reaction results. The experimental results are shown in Table 9:

[0170] Table 9. Anti-E (Fab) agglutination inhibition test results

[0171]

[0172] The above experimental results show that the antibodies in the tested serum are inhibited by anti-E (Fab), indicating that the antibody specificity is consistent with anti-E, that is, the antibodies in the tested serum are anti-E.

[0173] 6. Verification of antibody specificity identification results

[0174] In order to further confirm the antibody specificity identification results, combined with the sample conditions and laboratory reality, it was decided to use the spectrum cells with batch number 20201113 produced by Shanghai Blood Biotechnology Co., Ltd. for further antibody identification. The identification method used the standard spectrum cell microcolumn gel method with higher sensitivity.

[0175] (1) Label Diameca and dilute the commercially available spectrum cells numbered 1-10 with diluent No. 2 that has been equilibrated at room temperature to a final concentration of 0.8%.

[0176] (2) Add 50 μl of the diluted spectrum cells numbered 1-10 to the marked diamica columns, and then add 25 μl of the subject's plasma.

[0177] (3) Incubate at 37°C for 15 minutes.

[0178] (4) Centrifuge for 10 minutes using the matching centrifuge and record and interpret the analysis results.

[0179] The experimental results further confirmed that the antibody contained in the patient's serum was anti-E, see Table 10.

[0180]

[0181] The results of the internal quality control and external quality assessment of this laboratory showed that no out-of-control problems occurred, and the negative and positive control results of each part of the experiment were normal. After comparative analysis, the subject was anti-E, and the presence of other antibodies in the plasma was not completely ruled out. The blood matching method and steps were strictly carried out in accordance with the standard operating procedures of the Institute of Transfusion Medicine, and finally the blood donors with the same phenotype as the patient were selected for blood distribution.

[0182] Example 3. A case of anti-M found in a blood donor's ABO blood type test

[0183] On December 28, 2020, we received a case of ABO blood typing sent to the laboratory. The blood donation code of the test specimen was 0080120031492 (X-20-986). It was suspected to be anti-M. The identification process is as follows:

[0184] 1. Sample reception and preprocessing

[0185] (1) The experimenter reviews the blood sample and the test submission form, confirms that the sample status meets the requirements, receives the sample, and fills in the relevant records.

[0186] (2) Prepare experimental reagents and equipment, and check whether the experimental equipment is operating normally.

[0187] (3) Separation of plasma and red blood cells: Centrifuge the anticoagulated blood and separate it. Use a pipette to draw out the subject's plasma and put it into a clearly labeled test tube for later use.

[0188] (4) Preparation of red blood cells for the subject's experiment: Take 2 to 3 drops of anticoagulated red blood cells, wash them three times with normal saline, and prepare a 2% to 5% red blood cell suspension (1 volume of packed red blood cells plus 16 to 40 volumes of normal saline) for later use.

[0189] 1. ABO blood typing (test tube method)

[0190] Positive typing: Take 4 clean test tubes and label them with anti-A, anti-B, anti-AB and anti-H respectively. Use a dropper to add 1 drop of anti-A, anti-B, anti-AB and anti-H typing serum to the bottom of the test tubes respectively. Then use a dropper to add 1 drop of the subject's 2%-5% red blood cell suspension respectively. Mix well. Centrifuge at 900-1000g for 15 seconds and observe the results.

[0191] Reverse typing: Take another 4 clean test tubes and label them as A cells, B cells, O cells and self cells. Use a dropper to add 2 drops of the subject's plasma to each of the above labeled test tubes. Then use a dropper to add 1 drop of A, B, O reverse typing red blood cell suspension and 2%-5% red blood cell suspension of the subject, respectively, and mix well. Centrifuge at 900-1000g for 15 seconds and observe the results.

[0192] 2. Antibody Screening (Saline Method)

[0193] (1) Take 4 test tubes and number them 1, 2, 3, and autologous. Add 2 drops of the subject's plasma to each of the 4 test tubes and 1 drop of screening cells I, II, III and autologous red blood cells respectively. Mix well and incubate at room temperature for 10-15 minutes. Centrifuge at 900-1000g for 15 seconds to observe whether hemolysis occurs. Gently resuspend the cells and observe the agglutination reaction. Record the results of the room temperature saline method.

[0194] (2) Place the test tube in a 37°C water bath for 30 minutes. Centrifuge at 900-1000 g for 15 seconds to observe whether there is hemolysis and agglutination, and record the results of the 37°C saline method.

[0195] 3. Antibody screening (gel card method)

[0196] (1) Prepare I, II, and III standard antibody screening cells (0.8%), equilibrate diluent No. 2 at room temperature, prepare samples and label Diameka.

[0197] (2) Add 50 μl of antibody screening I, II, and III cells to each of the three tubes, and add 25 μl of the subject's plasma. Incubate at 37°C for 15 minutes.

[0198] (3) Use the matching centrifuge to centrifuge for 10 minutes and read and record the results. The experimental results are shown in Table 11:

[0199] Table 11. Antibody screening (gel card method) results

[0200]

[0201] Note: Beijing refers to the standard serum and red blood cells produced by Beijing Jinhao Pharmaceutical Co., Ltd.; Changchun refers to the standard serum and red blood cells produced by Changchun Bode Biotechnology Co., Ltd.

[0202] The results showed that the results of the red blood cell typing test were inconsistent with the serum typing test. When the subject's own red blood cells and plasma did not agglutinate, the subject's plasma and A, B, and O reverse-typed red blood cells generally agglutinated, but the agglutination intensity of A1c and the subject's plasma was the highest, reaching 3+. And at 37°C, the agglutination intensity of the subject's plasma and B and O reverse-typed red blood cells was significantly weakened. Anti-screening was positive, revealing the presence of the same type of saline room temperature antibodies other than the ABO system in the subject's plasma.

[0203] Based on past experience, most antibodies in saline and room temperature are of the MNSs system. After further testing, the phenotype of the red blood cells of the subjects was NNSs, so it was preliminarily inferred that this antibody was natural anti-M.

[0204] 4. Antibody specificity identification (anti-M (Fab) agglutination inhibition test method)

[0205] (1) Take one drop of type O red blood cells with a known phenotype of MM, wash them three times with 0.9% saline, and prepare a 2% to 5% red blood cell suspension.

[0206] (2) Take two small test tubes, one marked as Fab and the other as control. Add 1 drop of the above 2% to 5% red blood cell suspension to each of the two test tubes, 2 drops of anti-M (Fab) to the Fab tube, and 2 drops of 0.9% saline to the control tube, mix thoroughly, and allow to react at room temperature for 15 minutes.

[0207] (3) Wash the cell suspensions in the two test tubes three times with 0.9% saline and finally add 1 drop of saline to each tube to make a 2% to 5% suspension.

[0208] (4) Add 2 drops of serum to be tested to each of the two test tubes, mix thoroughly, centrifuge at 900-1000 g for 15 seconds, observe whether hemolysis occurs, gently resuspend the cells, observe the agglutination reaction, and record the results of the room temperature saline method.

[0209] The experimental results are shown in Table 12.

[0210] Table 12. Results of anti-M (Fab) agglutination inhibition assay

[0211]

[0212] The above experimental results show that the antibodies in the tested serum are inhibited by anti-M (Fab), indicating that the antibody specificity is consistent with anti-M, that is, the antibodies in the tested serum are anti-M.

[0213] 5. Verification of antibody specificity identification results

[0214] To further prove the accuracy of the experimental results, combined with the sample conditions and laboratory reality, the spectrum cells with batch number 20201113 produced by Shanghai Blood Biotechnology Co., Ltd. were used for further antibody identification, and the identification method used the microcolumn gel method with higher sensitivity.

[0215] (1) Label Diameca and dilute the commercially available spectrum cells numbered 1-10 with diluent No. 2 that has been equilibrated at room temperature to a final concentration of 0.8%.

[0216] (2) Add 50 μl of the diluted spectrum cells numbered 1-10 to the marked diamica columns, and then add 25 μl of the subject's plasma.

[0217] (3) Incubate at 37°C for 15 minutes.

[0218] (4) Centrifuge for 10 minutes using a matching centrifuge, and record and interpret the analysis results. The experimental results are shown in Table 13.

[0219]

[0220] 6. Further identification of MN blood type

[0221] Take two small test tubes, mark them as anti-M and anti-N, add 1 drop of anti-M and anti-N serum, then add 1 drop of 2% to 5% red blood cell suspension of the subject, mix gently, leave at room temperature for 1 minute, centrifuge at 900 to 1000g for 15 seconds, observe and record the results.

[0222] The results of MN blood typing are shown in Table 14:

[0223] Table 14MN blood type identification results

[0224]

[0225] The experimental results showed that the subject's red blood cells only agglutinated with the anti-N reagent, and could be determined to be of NN blood type.

[0226] The subject's plasma showed general agglutination with B and O reverse-typed red blood cells, and anti-M was found in his plasma. Therefore, the experimenter decided to perform MN blood type identification on B and O reverse-typed red blood cells. The experimental method is the same as step 6. Further identification of MN blood type. The results of this part of the experiment showed that B and O reverse-typed red blood cells and anti-M typing serum can produce agglutination under test tube conditions. This shows that there is M antigen on the surface of B and O reverse-typed red blood cells, which can react with anti-M in the subject's plasma under room temperature. The results of the laboratory's internal quality control and inter-laboratory quality assessment showed that no out-of-control problems occurred, and there were no abnormalities in the negative and positive control results of each part of the experiment. Therefore, the presence of alloantibodies in the subject's plasma is the main reason for the discrepancy between the positive and negative typing of the subject under room temperature conditions.

[0227] Through comprehensive comparative analysis of each part of the experiment, the subject was found to be type B, with anti-M alloantibodies in plasma, which does not completely rule out the presence of other antibodies in plasma.

Claims

1. A method for specific identification of irregular blood type antibodies, comprising the following steps: (a) preparing blood type specific antibody Fab fragments; (b) Identification of the specificity of incomplete antibodies, Wherein step (a) further comprises taking specific blood type antibody IgG to prepare the specific blood type antibody Fab fragment; Step (b) further comprises: (1) Irregular antibodies are found; the irregular antibodies are found in the blood during cross-matching, antibody screening, neonatal hemolytic disease test and transfusion reaction test; (2) Preliminarily infer the category of the specificity of irregular antibodies; preliminarily infer the range of antibody specificity based on the blood type serological characteristics of the antibodies; (3) using the specific blood type antibody Fab fragment prepared in step (a) to prepare sensitized red blood cells; using the Fab fragment of the irregular antibody that is most likely to be derived from sub-step (2) to co-incubate with red blood cells known to be positive for the corresponding antigen, and fully sensitize them under specific temperature, ionic strength, and pH conditions; then washing and preparing a 2% to 5% cell suspension for later use; (4) reacting the test serum with sensitized red blood cells or red blood cells positive for the corresponding antigen; taking the test serum and a 2% to 5% suspension of the corresponding sensitized red blood cells, reacting them in saline, enzyme medium, polybrene medium or anti-human globulin medium at 2°C to 39°C, and performing a parallel control experiment with the test serum and red blood cells positive for the corresponding antigen; (5) Determination of results: ① When the test serum agglutinates with the control cells but not with the sensitized red blood cells, it indicates that the corresponding antigen on the red blood cells is blocked by Fab, and the specificity of the antibody in the test serum is consistent with the specificity of Fab; ② When the test serum agglutinates with the control experiment but also with the sensitized red blood cells, there are two possibilities: one is that the antibody specificity of the test serum is inconsistent with the specificity of the Fab fragment; the other is that the test serum contains more than two complex antibodies, which requires further identification, that is, sensitizing red blood cells with different specific Fab combinations, observing and analyzing the results, and making a judgment; ③ When the control experiment does not agglutinate, it indicates that there is an error in the experimental process and the experimental results are unreliable. It is necessary to recheck the experimental conditions or replace appropriate control red blood cells and repeat the experiment.

2. The method for specific identification of irregular blood type antibodies according to claim 1, wherein the specific blood type antibody IgG in step (a) is anti-D, anti-E, anti-C, anti-E, or anti-K.

3. The method for specific identification of irregular blood type antibodies according to claim 1, wherein the specific blood type antibody IgG in step (a) can be a self-prepared polyclonal antibody or monoclonal antibody, or a commercially available blood type reagent.

4. The method for specific identification of irregular blood type antibodies according to claim 1, wherein sub-step (2) in step (b) further comprises inferring the specificity of the irregular antibodies by examining the relevant antigens of the tested red blood cells.

5. The method for specific identification of irregular blood type antibodies according to claim 1, wherein for the red blood cell ccdee phenotype, the irregular antibodies with greater possibility are anti-D, anti-E and anti-C, and the anti-D Fab fragment is selected to be co-incubated with D antigen-positive red blood cells, the anti-E Fab fragment is co-incubated with E antigen-positive red blood cells, or the anti-C Fab fragment is co-incubated with C antigen-positive red blood cells to prepare the sensitized red blood cells.

6. The method for specific identification of irregular blood type antibodies according to claim 1, wherein sub-step (4) in step (b) comprises taking the test serum and a 2% to 5% suspension of the corresponding sensitized red blood cells, reacting them in saline, enzyme medium, polybrene medium or anti-human globulin medium at 4°C, respectively, and performing a parallel control experiment with the test serum and the corresponding antigen-positive red blood cells.

7. The method for specific identification of irregular blood type antibodies according to claim 1, wherein sub-step (4) in step (b) comprises taking the test serum and a 2% to 5% suspension of the corresponding sensitized red blood cells, reacting them in saline, enzyme medium, polybrene medium or anti-human globulin medium at 22°C, respectively, and performing a parallel control experiment with the test serum and the corresponding antigen-positive red blood cells.

8. The method for specific identification of irregular blood type antibodies according to claim 1, wherein sub-step (4) in step (b) comprises taking the test serum and a 2% to 5% suspension of the corresponding sensitized red blood cells, reacting them in saline, enzyme medium, polybrene medium or anti-human globulin medium at 37°C, respectively, and performing a parallel control experiment with the test serum and the corresponding antigen-positive red blood cells.

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