An IgM antibody for preventing erythrocyte agglutination, its preparation method and uses

Abstract

This invention relates to the field of biological detection, and in particular to an IgM antibody for preventing erythrocyte agglutination, its preparation method, and its uses. The IgM antibody for preventing erythrocyte agglutination is obtained by mixing and incubating a natural IgM antibody with DTT. The IgM antibody can be used to prepare products for detecting ABO blood types and products for detecting the expression levels of type A antigen and / or type B antigen. The prepared products can be used for quantitative detection of ABO antigen expression, studying changes in ABO antigen expression levels among different genotypes, and studying diseases related to ABO antigen expression levels.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and in particular to an IgM antibody for preventing erythrocyte agglutination, its preparation method, and its uses. Background Technology

[0002] The amount of red blood cell antigens is crucial for blood type-related immune responses. Typically, fluorescently labeled IgG antibodies are used to quantitatively detect antigens; IgG is quantified by detecting fluorescence intensity, and antigen expression is quantified based on the amount of IgG antibody binding. However, in studies of ABO antigens, both IgG-A and -B can cause strong agglutination of red blood cells, making it difficult to detect antigen expression levels on individual cells. Currently, a common method is to use aldehyde-treated red blood cells to inhibit cell agglutination for flow cytometry detection; however, this method is influenced by many factors and exhibits significant batch-to-batch variability. Because aldehyde treatment alters the properties of the red blood cell membrane, the detection data may not fully represent the actual antigen expression level.

[0003] The study of ABO blood group antigen expression levels is of great significance. Blood group antigen levels exhibit significant individual variability; for example, there is a marked difference in expression levels between homozygous and heterozygous ABO gene individuals. Furthermore, various diseases can lead to changes in ABO antigen levels. For instance, ABO antigens are weakened in hematological malignancies, particularly acute myeloid leukemia (AML). This weakening often precedes the clinical diagnosis of leukemia, and the underlying mechanism is generally believed to be the high level of recombination activity of the ABO gene in leukemia patients. The expression levels of erythrocyte blood group antigens may also change in infants with hemolytic disease of the fetus and newborn (HDFN). In addition, the expression and changes of erythrocyte antigens have a crucial impact on the quality of reagent cells. Deterioration in antigen expression on reagent cells, or weakening of antigens due to storage, will severely affect the accuracy of immunohematological tests and the reliability of internal quality control. During storage, the intensity of reagent erythrocyte antigens gradually decreases. By quantifying blood group antigens, suitable donor red blood cell samples can be selected, the shelf life of reagent red blood cells can be determined, and red blood cell quality control products with constant antigen expression levels can be prepared.

[0004] Methods for quantitative detection of blood group antigens include radioimmunoassay using labeled antibodies or exogenous lectins, electron microscopy, and flow cytometry. Among these, the quantitative detection method combining flow cytometry and monoclonal antibody technology is currently the most advanced. Flow cytometry can analyze tens of thousands of cells at the cellular and molecular level using monoclonal antibodies at high speed, and can simultaneously measure multiple parameters from a single cell, offering advantages such as speed, high precision, and good accuracy. Flow cytometry is very common in quantitative red blood cell blood typing studies, such as the quantitative study of blood group antigens in the Kell and Rh systems.

[0005] Because flow cytometry requires single-cell suspensions as test samples, ABO blood group antigens, which are easily agglutinated by antibodies, cannot be directly detected by flow cytometry. Previously, to apply flow cytometry to the detection of ABO antigens, Berneman ZN et al. treated red blood cells with dimethyl nitrite (DMS), formaldehyde, or glutaraldehyde (GA) and prepared low-affinity FITC-IgG Fab fluorescent antibodies to label the aldehyde-treated red blood cells to assess the amount of AB antigen. They used this new method to detect higher AB antigen expression levels than previous radioimmunoassays. However, their research also mentioned the limitations of this red blood cell fixation method: when the amount of glutaraldehyde is low, cell clots increase; when the amount of glutaraldehyde is high, background values ​​increase and detection values ​​decrease, leading to increased error. Red blood cells with higher antigen expression levels are prone to agglutination, and flow cytometry, to ensure the accuracy of single-cell mean measurements, excludes these agglutinated, high-expressing cells, resulting in measurement errors. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide an IgM antibody for preventing erythrocyte agglutination, its preparation method and uses, in order to solve the problems in the prior art.

[0007] To achieve the above and other related objectives, the present invention first provides an IgM antibody for preventing erythrocyte agglutination, wherein the IgM antibody is obtained by mixing and incubating a natural IgM antibody with DTT.

[0008] The present invention also provides a method for preparing the IgM antibody, the method comprising the following steps: mixing DTT solution with natural IgM antibody and incubating to obtain the IgM antibody for preventing erythrocyte aggregation.

[0009] The present invention also provides a product for preventing erythrocyte agglutination, the product comprising the IgM antibody and iodoacetamide.

[0010] The present invention also provides a method for preparing the product, the method comprising: mixing iodoacetamide with the IgM antibody to obtain the product.

[0011] The present invention also provides the use of the IgM antibody and the product in the preparation of products for detecting ABO blood group and / or in the preparation of products for detecting the expression levels of type A antigen and / or type B antigen.

[0012] As described above, the IgM antibody for preventing erythrocyte agglutination, its preparation method, and its uses according to the present invention have the following beneficial effects:

[0013] 1) The prepared IgM antibody and / or product for preventing red blood cell aggregation can be used for direct quantitative detection of ABO blood group A antigen and / or B antigen expression in healthy individuals, detection of red blood cell products using routine ABO blood group reagents, study of changes in ABO antigen expression levels under different physiological conditions, and study of diseases related to ABO antigen expression levels.

[0014] 2) Compared with traditional methods, the antibody / product prepared in this invention for preventing red blood cell agglutination is simple to operate and has small batch-to-batch differences when detecting the expression levels of type A antigen and / or type B antigen. It can be used for large-scale detection of ABO blood type and the expression levels of type A antigen and / or type B antigen. Attached Figure Description

[0015] Figure 1 The diagram illustrates the ability of different concentrations of DTT to treat antibodies against blood type A and B to label ABO antigens according to the present invention. Figure 1 .

[0016] Figure 2 The diagram illustrates the ability of different concentrations of DTT to treat antibodies against blood type A and B to label ABO antigens according to the present invention. Figure 2 .

[0017] Figure 3 The diagram illustrates the ability of different concentrations of DTT to treat antibodies against blood type A and B to label ABO antigens according to the present invention. Figure 3 .

[0018] Figure 4 The diagram shows the flow cytometry results of anti-A blood group antigen antibody treated with 1mM DTT for different number of days to detect A antigen. (A) The diagram shows the flow cytometry of anti-A blood group antigen antibody treated with 1mM DTT at 4℃ for 7 days; (B) The diagram shows the flow cytometry of anti-A blood group antigen antibody treated with 1mM DTT at 4℃ for 3 days. Detailed Implementation

[0019] The present invention first provides an IgM antibody for preventing erythrocyte aggregation, wherein the IgM antibody is obtained by mixing and incubating a natural IgM antibody with DTT.

[0020] In some embodiments of the present invention, the natural IgM antibody is an anti-A and / or anti-B antigen antibody.

[0021] Furthermore, the anti-A and anti-B antigen antibodies are anti-A and anti-B blood typing reagents.

[0022] Furthermore, the anti-A and anti-B blood typing reagents are commercially available reagents.

[0023] In this invention, the concentration of DTT is determined by evaluating the ability of IgM antibody to label antigen. The working concentration of DTT is obtained through the following steps: preparing DTT diluents at different dilution factors, mixing each DTT diluent with natural IgM antibody and incubating, and after incubation, continuing to incubate the incubated system with type A antigen and / or type B antigen. After incubation, the working concentration of DTT is determined based on the concentration of the DTT diluent used in the group where no agglutination occurred.

[0024] In some embodiments of the present invention, the working concentration of DTT refers to the final concentration of DTT diluent in the mixture after mixing the natural IgM antibody with DTT diluent, based on the volume of the mixture.

[0025] In some embodiments of the present invention, "no agglomeration" means that the agglomeration result is 1+w, ±, or θ.

[0026] In some embodiments of the present invention, the type A antigen and / or type B antigen may also be type A and / or type B erythrocytes.

[0027] In some embodiments of the present invention, the ratio of the reciprocal of the titer of the natural IgM antibody to the working concentration of DTT is 800:0.25-5mM.

[0028] In a preferred embodiment of the present invention, the ratio of the reciprocal of the titer of the natural IgM antibody to the working concentration of DTT is 800:0.5-2mM.

[0029] In a more preferred embodiment of the present invention, the ratio of the reciprocal of the natural IgM antibody titer to the working concentration of DTT is 800:0.5-1.5 mM. The ratio of the reciprocal of the natural IgM antibody titer to the working concentration of DTT is selected from any of the following ranges: 800:0.5-0.7 mM, 800:0.7-0.9 mM, 800:0.9-1.1 mM, 800:1.1-1.3 mM, 800:1.3-1.5 mM.

[0030] In a preferred embodiment of the present invention, the ratio of the reciprocal of the titer of the natural IgM antibody to the working concentration of DTT is 800:1mM.

[0031] In this invention, the properties of erythrocytes are not altered; only the reactivity of the antibody is changed. Utilizing the strong reducing properties of DTT, erythrocyte agglutination is avoided without affecting antibody specificity. DTT can disrupt the SS bonds between IgM chains. This method involves adding DTT to natural IgM to treat 19S IgM polymeric antibodies into 9S IgM monomers.

[0032] In this invention, the IgM antibody can be used alone, but if the IgM antibody is used alone, it needs to be prepared fresh for each use.

[0033] The present invention also provides a method for preparing the IgM antibody, the method comprising the following steps: mixing DTT solution with natural IgM antibody and incubating to obtain the IgM antibody for preventing erythrocyte aggregation.

[0034] In some embodiments of the present invention, the DTT solution is obtained by mixing DTT with a buffer solution.

[0035] In a preferred embodiment, the buffer solution is PBS with a pH of 7.4.

[0036] The working concentration of DTT is obtained through the following steps: preparing DTT diluents at different dilution ratios, mixing each DTT diluent with natural IgM antibody and incubating, and after incubation, continuing to incubate the incubated system with type A antigen and / or type B antigen. After incubation, the working concentration of DTT is determined based on the concentration of the DTT diluent used in the group where no agglutination occurred.

[0037] In some embodiments of the present invention, the ratio of the reciprocal of the titer of the natural IgM antibody to the working concentration of DTT is 800:0.25-5mM.

[0038] In a preferred embodiment of the present invention, the ratio of the reciprocal of the titer of the natural IgM antibody to the working concentration of DTT is 800:0.5-2mM.

[0039] In a more preferred embodiment of the present invention, the ratio of the reciprocal of the natural IgM antibody titer to the working concentration of DTT is 800:0.5-1.5 mM. The ratio of the reciprocal of the natural IgM antibody titer to the working concentration of DTT is selected from any of the following ranges: 800:0.5-0.7 mM, 800:0.7-0.9 mM, 800:0.9-1.1 mM, 800:1.1-1.3 mM, 800:1.3-1.5 mM.

[0040] In a preferred embodiment of the present invention, the ratio of the reciprocal of the titer of the natural IgM antibody to the working concentration of DTT is 800:1mM.

[0041] In some embodiments of the present invention, the incubation temperature is 2 to 6°C.

[0042] In a preferred embodiment of the present invention, the incubation temperature is 4°C.

[0043] In some embodiments of the present invention, the incubation time is 24 hours to 7 days.

[0044] In a preferred embodiment of the present invention, the incubation time is 3 to 7 days.

[0045] In a more preferred embodiment of the present invention, the incubation period is 7 days.

[0046] The present invention also provides a product for preventing erythrocyte agglutination, the product comprising the IgM antibody and iodoacetamide.

[0047] In this invention, if the IgM antibody needs to be stored after preparation, it can be mixed with iodoacetamide to prepare the product used to prevent erythrocyte aggregation. Iodoacetamide is used to block free thiol groups. Since DTT has two thiol groups, it can reduce two disulfide bonds, thus requiring a reaction with two iodoacetamide molecules. In this invention, the amount of iodoacetamide in the product is determined by the amount of DTT added during the preparation of the IgM antibody.

[0048] In some embodiments of the invention, the iodoacetamide is in excess of DTT. The amount of DTT refers to the amount of DTT added during the preparation of the IgM antibody.

[0049] In some embodiments of the present invention, the molar ratio of iodoacetamide to DTT is 1:2 to 3.

[0050] In a preferred embodiment of the present invention, the molar ratio of iodoacetamide to DTT is 1:2.5.

[0051] In some embodiments of the present invention, the CAS number of the iodoacetamide is 144-48-9.

[0052] Iodoacetamide is mixed with the DTT-treated IgM antibody. Iodoacetamide can block the thiol group, thereby maintaining the stability of the IgM monomer. After this treatment, the IgM antibody will not cause erythrocyte agglutination and will have the ability to quantitatively detect antigens.

[0053] The present invention also provides a method for preparing the product, the method comprising: mixing iodoacetamide with the IgM antibody to obtain the product.

[0054] The present invention also provides the use of the IgM antibody and the product in the preparation of products for detecting ABO blood group and / or products for detecting the expression levels of type A antigen and / or type B antigen.

[0055] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0056] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0057] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0058] Example 1: Preliminary confirmation of optimal reaction conditions

[0059] 1.1 Reagent Preparation

[0060] Prepare a dithiothreitol (DTT) solution using 0.1M, pH 7.4 PBS. Weigh the DTT powder and prepare a 200mM DTT solution using PBS. Then dilute the prepared 200mM DTT solution with PBS to 2mM / 10mM / 20mM / 40mM, and prepare 1 mL of each solution.

[0061] 1.2 Antibody treatment

[0062] Mix 450 μL of DTT diluent with 450 μL of anti-A / anti-B blood typing reagent (the titer of the -A / -B reagent is 1:800) in equal volumes, and mix thoroughly. The concentrations of DTT in the resulting -A / -B reagents are 1 mM / 5 mM / 10 mM / 20 mM, respectively.

[0063] 1.3 Set different reaction conditions

[0064] 900 μL of the DTT and -A / -B mixture was aliquoted into two EP tubes, labeled 4℃ and room temperature (25℃), respectively. 400 μL of DTT was added to each EP tube to treat the -A / -B reagent.

[0065] 1.4 Comparison of the cell agglutination effect of reagents at different reaction times

[0066] At intervals of 1 h, 2 h, 4 h, 24 h, 48 h, and 72 h, 50 μL of DTT treatment-A / -B reagent was taken from the EP tube and added to a small test tube. Then, 50 μL of type A / B reagent red blood cells were added. After incubation at room temperature for 15 min, the tubes were centrifuged and the results were observed. The experimental results are shown in Table 1.

[0067] Table 1. Effects of different concentrations of DTT on the disruption of A / B cell aggregation under 4℃ and room temperature conditions.

[0068]

[0069]

[0070] Note: 4+ represents a very strong agglutination reaction, where almost all cells form agglutination clumps; 3+ represents a strong agglutination reaction, where most cells form agglutination clumps; 2+ represents a moderate agglutination reaction, where some cells form agglutination clumps; 1+ represents a weak agglutination reaction, where only a small number of cells form agglutination clumps; ± represents very weak agglutination, characterized by a small number of cell clumps when the test tube is gently shaken, but the clumps disappear with continued shaking; s represents "strong," indicating agglutination slightly stronger than the numerically labeled agglutination, for example, "1+s" means slightly stronger than "1+," but less intense than the "2+" range; w represents "weak," indicating agglutination slightly weaker than the numerically labeled agglutination, for example, "1+w" means slightly weaker than "1+," but more intense than the "±" range; θ represents no agglutination reaction, where no cells form agglutination clumps.

[0071] According to Table 1, DTT concentrations above 1 mM and incubation at room temperature for 24 h, and DTT concentrations above 2 mM and incubation at 4°C for 48 h, can prevent A / B antibody agglutination of red blood cells. At 72 h, A / B antibodies did not agglutinate red blood cells at any of the four DTT concentrations.

[0072] 1.5 Streaming Validation

[0073] 20 μL of -A / -B reagent treated with 1 mM / 5 mM / 10 mM / 20 mM DTT for 72 hours at room temperature / 4℃ was added to a small test tube and mixed thoroughly with 10 μL of type A / B erythrocytes. The mixture was incubated at 4℃ for 1 h, washed three times with ice-cold PBS, and then 1:500 diluted anti-mouse IgM-μ chain APC fluorescent secondary antibody was added. The mixture was incubated at 4℃ for 0.5 h, washed three times with ice-cold PBS, and resuspended to 800 μL for flow cytometry detection.

[0074] The flow cytometry results are shown in Table 2. The reaction conditions that resulted in the highest -A detection value were a DTT concentration of 1 mM and treatment at 4°C for 72 h; the reaction conditions that resulted in the highest -B detection value were a DTT concentration of 1 mM and treatment at 25°C for 72 h. Higher -A and -B detection values ​​indicate a stronger ability of the reagent to label erythrocyte ABO antigens.

[0075] Table 2. Labeling effect of different concentrations of DTT after treatment with reagents A and B by flow cytometry.

[0076]

[0077] 1.6 Preliminary Conclusions

[0078] According to Table 2 in section 1.6, the lower the DTT concentration, the stronger the labeling ability of the -A / -B reagent, and the effect of treating the -A / -B reagent antibody at 4℃ is better than that of treating the antibody at room temperature. Among them, the effect of treating the -A / -B reagent with 1mM DTT at 4℃ is the best. To further explore the optimal DTT concentration and reaction conditions, this study continued to adjust the DTT concentration and extend the reaction time.

[0079] Example 2 further investigates the optimal reaction concentration and reaction time of DTT.

[0080] 2.1 Reagent preparation and reaction conditions

[0081] Following the methods in 1.1-1.3, prepare DTT treatment-A / -B reagents of 0.1mM / 0.25mM / 0.5mM / 1mM / 5mM / 10mM / 20mM respectively, and react them at room temperature and 4℃ respectively.

[0082] 2.2 Cell agglutination effect of reagents at different reaction times

[0083] At 24h / 72h / 168h (i.e., 1 day, 3 days, and 7 days), 50μL of DTT treatment-A / -B reagent was taken from the EP tube and added to a small test tube, followed by 50μL of type A / B reagent red blood cells. After incubation at room temperature for 15min, the mixture was centrifuged and the results were observed. The results of 0.1mM / 0.25mM / 0.5mM / 1mM DTT treatment for 1 day, 3 days, and 7 days are shown in Table 3. The results of 5mM / 10mM / 20mM treatment for 1 day and 3 days are consistent with those in 1.4. At 7 days of treatment, none of the reagents agglutinated red blood cells.

[0084] Table 3. Effects of different concentrations of DTT on the disruption of A / B cell aggregation under 4℃ and room temperature conditions.

[0085]

[0086] Note: The meanings of the symbols “4+”, “3+”, “2+”, “1+”, “s”, “w”, “±”, and “θ” are the same as those in Table 1.

[0087] 2.3 Flow Validation

[0088] According to Table 3, under room temperature conditions, the agglutination ability of the -A / -B reagent was weak after treatment with 0.25mM / 0.5mM / 1mM DTT for 3 days. Under 4℃ conditions, the agglutination ability was weak after treatment with -A / -B reagent for 7 days with DTT concentrations ≥1mM. A DTT concentration of 0.1mM could not destroy the antibody's agglutination ability. To investigate the optimal DTT concentration and reaction time for antibody treatment, -A / -B reagent antibodies treated with 0.25mM / 0.5mM / 1mM DTT for 3 days at room temperature and -A / -B reagent antibodies treated with 1mM / 5mM / 10mM / 20mM DTT for 7 days at 4℃ were selected. The method was the same as in 1.5, and the ABO antigen labeling effect of DTT-treated antibodies was detected. The results are shown in Table 4.

[0089] Table 4. Flow cytometry detection of the effect of DTT-treated antibody-labeled ABO antigen 2

[0090]

[0091] Example 3: Determining the optimal DTT treatment conditions for reagents A and B.

[0092] Based on the flow cytometry results in Table 2, plotting can be performed. Figure 1 The conditions were incubation at room temperature and 4°C for 3 days. The flow cytometry results were plotted in conjunction with Tables 2 and 4. Figure 2 and Figure 3 .exist Figure 2 In the diagram, -1 and -2 represent the results of flow cytometry validation in section 1.5 and flow cytometry detection in section 2.3, respectively. Both flow cytometry validation in section 1.5 and flow cytometry validation in section 2.3 include the results of treating -A / -B antibodies with 1mM DTT for 3 days at room temperature, which are represented by 1mM-1 and 1mM-2.

[0093] in accordance with Figure 1 , Figure 2 At both 4°C and room temperature, with a DTT concentration of 1 mM, DTT treatment of -A / -B antibodies showed the best effect. The effect of treatment at 4°C was better than that at room temperature, which may be because DTT is gentler on IgM at 4°C. Figure 2In addition to 1 mM-1, the flow cytometry results of -A / -B are similar at the same DTT concentration, but the detection value of -A in 1 mM-1 may be too high.

[0094] in accordance with Figure 3 When the DTT concentration is 1 mM, both -A and -B reagents showed stronger ability to label ABO antigens after treatment at 4°C for 3 days. However, after treatment at 4°C for 7 days, the ability of -A and -B reagents to label ABO antigens was similar. Under these conditions, the antigen labeling effect of this reagent may be more stable, and the flow cytometry ( Figure 4 As shown in Figure A, taking -A as an example, the horizontal axis of both Figures A and B represents forward scattered light (FSC), and the vertical axis represents side scattered light (SSC). The large cell cluster in the center of the figure represents the target cell cluster, which should be a single-cell suspension. If cell clots are present in the sample, small cell clusters will appear to the upper right of the central cell cluster. Figure B shows significantly more agglutinated cells than Figure A. Therefore, at a DTT concentration of 1 mM, a 7-day reaction at 4°C results in less antibody-induced cell agglutination compared to a 3-day reaction at 4°C. Further increasing the reaction time is meaningless, as it leads to DTT inactivation.

[0095] Example 4 determines the preparation method of the flow cytometry reagent for detecting high expression of blood group antigens.

[0096] The appropriate reaction conditions were selected as follows: a DTT concentration of 1 mM and treatment at 4°C for 7 days. A dithiothreitol (DTT) solution of 2 mM was prepared using 0.1 M PBS (pH 7.4). This 2 mM DTT solution was then thoroughly mixed with the -A / -BIgM monoclonal antibody reagent and incubated at 4°C for 7 days. To ensure reagent stability, excess iodoacetamide (IAA) was added to the -A / -B IgM reagent after incubation, bringing the final IAA concentration to 5 mM.

[0097] Example 5

[0098] 5.1 Reagents and Instruments

[0099] Gene extraction kit: batch number W0202, TIANGEN; Taq enzyme: batch number AL51581A, TaKaRa; E6 primer: batch number 112793654, Sangon Biotech; 0.1M, pH 7.2 PBS buffer: laboratory-made, concentration; anti-A and anti-B blood typing reagent: batch number 20220711, Shanghai Blood Biotechnology; -A direct labeling antibody: 9431PE, BGRL; -B direct labeling antibody: 9434PE, BGRL; goat anti-mouse IgM-μ chain APC fluorescent secondary antibody: batch number ab150123, Abcam; glutaraldehyde reagent: batch number 20150114, Sinopharm Chemical Reagent Co., Ltd.

[0100] GeneAmp PCR amplification instrument: Model 9700, Applied Biosystems. Curiox plate washer: HT 2000, Curiox. Laminar Wash TM 96-well plate: Lot number C96DAC6E24024, Curiox. Flow cytometer: BC36267, Beckman. U-bottom 96-well plate: 3799, Corning.

[0101] 5.2 Collect blood samples from ABO homozygous and heterozygous individuals from healthy individuals.

[0102] All homozygous AA, heterozygous AO, homozygous BB, and heterozygous BO samples were collected from 555 healthy blood donors. The E6 exons of type A and type B blood samples were amplified and sequenced (sent to Sangon Biotech for sequencing) to distinguish between AA / BB homozygotes and AO / BO heterozygotes (using Chromas software). Twenty homozygotes and 20 heterozygotes from type A and type B were collected, totaling 80 test results. The differences in A / B antigen expression levels were compared using the reagent antibody formulated in this patent and the traditional aldehyde method.

[0103] 5.3 The reagents formulated in this patent are used to detect the expression levels of A / B antigens in healthy individuals with homozygous / heterozygous ABO blood types.

[0104] Each blood sample was washed three times in 0.1M PBS buffer to prepare a 3% red blood cell suspension, which was then subjected to a laminar wash. TM Add 2.5 μL of red blood cell suspension to each well of a 96-well plate, followed by 5 μL of the reagent prepared in this patented formulation. Mix thoroughly and incubate at 4°C for 1 h. Wash 15 times with a Curiox washer, then add 1:500 diluted APC anti-mouse IgM μ-chain fluorescent secondary antibody, mix thoroughly, incubate in the dark for 30 min, wash 15 times with a Curiox washer, and transfer to a U-shaped 96-well plate with 200 μL of PBS added to each well for flow cytometry detection. All samples were tested in each experiment, and the experiment was independently repeated 3 times. The negative control consisted of red blood cell suspensions without the reagent prepared in this patented formulation, only APC anti-mouse IgM μ-chain fluorescent secondary antibody, with all other experimental conditions identical. The experiments were performed in parallel, and the results were recorded by flow cytometry. The experimental results were recorded as the mean fluorescence intensity (MFI) of the experimental group minus the MFI of the control group.

[0105] 5.4 Traditional aldehyde grading method for detecting A / B antigen expression levels in ABO homozygous / heterozygous ABO blood types in healthy individuals

[0106] After washing each blood sample three times with PBS and discarding the supernatant, collect the hematocrit red blood cells. Add 10 μL of hematocrit red blood cells to a small test tube, then add 330 μL of 0.01% glutaraldehyde solution (25% glutaraldehyde solution diluted 1:250 in PBS), shake to mix at room temperature for 10 minutes, centrifuge to remove the supernatant, and wash three times to prepare a 3% red blood cell suspension. Due to the short aldehydeation time, 10-12 red blood cell samples are aldehyde-treated each time.

[0107] Take 2.5 μL of 3% erythrocyte suspension from each tube and incubate with the PE-labeled fluorescent antibody. For type A erythrocytes, add 10 μL of -A-labeled antibody diluted 1:3.2 per aliquot; for type B erythrocytes, add 10 μL of -B-labeled antibody diluted 1:4 per aliquot. Both cells and PE-labeled fluorescent antibody are added to a laminar wash. TM In a 96-well plate, the mixture was thoroughly mixed and incubated at room temperature in the dark for 30 minutes. After washing 15 times with a Curiox plate washer, the mixture was transferred to a U-shaped 96-well plate with 200 μL of PBS added to each well. Flow cytometry was then performed. All samples were tested in each experiment, and the experiments were independently repeated three times. The negative control consisted of aldehyde-containing red blood cell suspensions without the addition of a direct-labeled fluorescent secondary antibody. All other experimental conditions were the same, and the experiments were performed in parallel. Flow cytometry results were recorded. The experimental results were recorded as the mean fluorescence intensity (MFI) of the experimental group minus the MFI of the control group.

[0108] 5.5 Comparison of detection results between the patented method and the traditional aldehyde conversion method

[0109] The results of three tests using this patented method and the traditional aldehyde conversion method are shown in Table 5. The mean values ​​were statistically analyzed, and a paired-samples t-test was performed for homozygotes and heterozygotes. A p-value less than 0.05 or 0.01 indicated a significant difference between homozygotes and heterozygotes; if a significant difference was observed, the specific difference was described by comparing the mean (or difference). Table 5 shows that eight paired data sets exhibited differences (p<0.05), including two groups of type B (groups 1 and 3) and two groups of type A (groups 2 and 3) in the aldehyde conversion method, and one group of type B (group 3) and three groups of type A (groups 1, 2, and 3) in the new method. In the aldehyde conversion method, among the three groups of type B, two groups had t-values ​​>0 and one group had t-values ​​<0, indicating that the t-values ​​in groups 2 showed that the expression level of BB homozygotes was higher than that of BO heterozygotes, and the t-value in group 1 showed that the expression level of BB homozygotes was lower than that of BO heterozygotes. In the aldehyde method, the t-values ​​for all three groups of type A were <0, indicating that the expression level of AA homozygotes was lower than that of AO heterozygotes. In the new method, the t-values ​​for all detection groups of types A and B were >0, indicating that the expression levels of A and B antigens in type A and type B homozygotes were higher than those in heterozygotes.

[0110] Table 5. Flow cytometry values ​​of ABO antigen expression levels in healthy individuals using the novel method and aldehyde method described in this patent.

[0111]

[0112] *p<0.05**p<0.01

[0113] The two methods yielded different conclusions. To verify which method was more accurate, the inter-batch differences between the two methods were compared using analysis of variance. The results are shown in Table 6.

[0114] A p-value less than 0.05 or 0.01 indicates a significant difference among the three groups. This suggests that in the aldehyde method, only the BB group (i.e., homozygous B-type individuals) showed no significant difference in detection values ​​across the three groups, with small inter-batch variations. In contrast, the BO / AA / AO groups showed larger inter-batch variations. The new method's p-values ​​were all >0.01, indicating small inter-batch variations in the results obtained using the new method.

[0115] The F-value is a statistical measure of the ratio of between-group differences to within-group differences. A larger F-value indicates a greater difference between groups relative to within-group differences, meaning the differences between different groups are more significant. The F-values ​​for the BO / AA / AO groups using the aldehyde method were all significantly larger than those for the new method. The F-values ​​for the new method were relatively stable.

[0116] Table 6. Results of Analysis of Variance

[0117]

[0118] *p<0.05**p<0.01

[0119] 5.6 Evaluation of Test Results

[0120] Based on the test results, the new method showed small batch-to-batch differences, indicating that its results are more reliable. It can be concluded that the expression levels of A / B antigens in homozygous A and B blood types are higher than in heterozygous individuals, suggesting that the expression levels of normal ABO blood group antigens are related to the ABO gene. Both the traditional method and the new detection system can detect ABO antigen expression levels in both homozygous and heterozygous ABO individuals; however, the aldehyde-treated erythrocyte method is complex, time-consuming, and yields unstable results with significant variations between different batches of erythrocytes. The newly established IgM antibody detection system has a simple experimental procedure and shows no significant inter-group differences.

[0121] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A mixture of IgM antibodies for preventing erythrocyte agglutination, characterized in that, The IgM antibody mixture is obtained by mixing and incubating natural IgM antibody with DTT. The natural IgM antibody is an anti-A antigen antibody and / or an anti-B antigen antibody. The ratio of the reciprocal of the titer of the natural IgM antibody to the working concentration of DTT is 800:0.25~5 mM.

2. The IgM antibody mixture according to claim 1, characterized in that, The working concentration of DTT is obtained through the following steps: preparing DTT diluents of different dilution ratios, mixing each DTT diluent with natural IgM antibody and incubating, and after incubation, continuing to incubate the incubated system with type A antigen and / or type B antigen. After incubation, the working concentration of DTT is determined based on the concentration of the DTT diluent used in the group where no agglutination occurs.

3. The IgM antibody mixture according to claim 1, characterized in that, The ratio of the reciprocal of the natural IgM antibody titer to the working concentration of DTT is 800:0.5~2 mM.

4. The method for preparing the IgM antibody mixture according to any one of claims 1 to 3, characterized in that, The preparation method includes the following steps: mixing DTT solution with natural IgM antibody and incubating to obtain the IgM antibody mixture for preventing erythrocyte aggregation.

5. The method for preparing the IgM antibody mixture according to claim 4, characterized in that, Includes one or more of the following conditions: 1) The working concentration of the DTT solution is obtained by the following steps: preparing DTT diluents of different dilution ratios, mixing each DTT diluent with natural IgM antibody and incubating, and after incubation, continuing to incubate the incubated system with type A antigen and / or type B antigen. After incubation, the working concentration of DTT is determined according to the concentration of the DTT diluent used in the group in which no agglutination occurs. 2) The DTT solution is obtained by mixing DTT with a buffer solution; 3) The ratio of the reciprocal of the titer of the natural IgM antibody to the working concentration of DTT is 800:0.25~5 mM; 4) The incubation temperature is 2~6℃; 5) The incubation period is 24 hours to 7 days.

6. The method for preparing the IgM antibody mixture according to claim 5, characterized in that, The ratio of the reciprocal of the natural IgM antibody titer to the working concentration of DTT is 800:0.5~2 mM; And / or, the incubation period is 3 to 7 days.

7. A product for preventing red blood cell agglutination, characterized in that, The product comprises the IgM antibody mixture of any one of claims 1 to 3 and iodoacetamide.

8. The product according to claim 7, characterized in that, The iodoacetamide is in excess compared to DTT.

9. The product according to claim 8, characterized in that, The molar ratio of iodoacetamide to DTT is 1:2~3.

10. The method for preparing the product according to any one of claims 7-8, characterized in that, The preparation method includes mixing iodoacetamide with the IgM antibody mixture according to any one of claims 1 to 3 to obtain the product.

11. Use of the IgM antibody mixture according to any one of claims 1 to 3 or the product according to any one of claims 7 to 8 in the preparation of products for detecting ABO blood group and / or the preparation of products for detecting the expression levels of type A antigen and / or type B antigen.

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