IgM (Immunoglobulin M) antibody for preventing erythrocyte agglutination as well as preparation method and application of IgM antibody

Abstract

The invention relates to the field of biological detection, in particular to an IgM antibody for preventing red blood cell agglutination and a preparation method and application thereof, and the IgM antibody for preventing red blood cell agglutination is obtained by mixing and incubating a natural IgM antibody and DTT. The IgM antibody can be used for preparing a product for detecting the ABO blood type and a product for detecting the expression quantity of an A-type antigen and / or a B-type antigen, and the prepared product can be used for quantitatively detecting the expression of the ABO antigen, researching the change condition of the expression quantity of different genotypes of ABO antigens, researching diseases related to the expression quantity of the ABO antigens and the like.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to an IgM antibody for preventing red blood cell agglutination, and a preparation method and application thereof. Background Art

[0002] The amount of blood type antigen on red blood cells is crucial to the immune response related to blood type. Usually, fluorescently labeled IgG antibodies can be used to quantitatively detect antigens, and IgG can be quantitatively detected by detecting the fluorescence intensity, and the antigen expression amount can be quantitatively detected based on the binding amount of IgG antibodies. However, when studying ABO antigens, since both IgG-A and -B can cause strong agglutination of red blood cells, it is difficult to detect the antigen expression amount on a single cell. The commonly used method currently uses aldehyde-treated red blood cells to inhibit cell agglutination for flow cytometry, but this method has many influencing factors and large batch differences. Because aldehyde changes the properties of the red blood cell membrane, the test data cannot fully represent the actual antigen expression amount.

[0003] The study of the expression of ABO blood type antigens is of great significance. There are large individual differences in the amount of blood type antigens, for example, there are significant differences in the expression of ABO gene homozygotes and heterozygotes. In addition, many diseases can cause changes in the amount of ABO antigens. For example, ABO antigens are weakened in malignant blood diseases, especially in acute myeloid leukemia. This antigen weakening often precedes the clinical diagnosis of leukemia. It is generally believed that its mechanism is that the ABO gene of leukemia patients has a high level of recombination activity. The expression of red blood cell blood type antigens in children with hemolytic disease of the fetus and newborn (HDFN) may also change. In addition, the expression and changes of red blood cell antigens have a critical impact on the quality of reagent cells. The deterioration of antigen expression on reagent cells, or the weakening of antigens due to storage, will seriously affect the accuracy of immunohematological testing and the reliability of in-house quality control. During storage, the strength of reagent red blood cell antigens will gradually weaken. By quantifying blood type antigens, it is possible to select appropriate donor red blood cell samples, determine the shelf life of reagent red blood cells, and prepare red blood cell quality control products with constant antigen expression levels.

[0004] Methods for quantitative detection of blood type antigens include radioimmunoassay using labeled antibodies or exogenous lectins, electron microscopy, and flow cytometry. Among them, the quantitative detection of antigens by combining flow cytometry with monoclonal antibody technology is currently the most advanced method. Flow cytometers can analyze tens of thousands of cells at high speed at the cellular molecular level through monoclonal antibodies, and can measure multiple parameters from one cell at the same time. It has the advantages of fast speed, high precision, and good accuracy. Flow cytometry is very common in the quantitative study of red blood cell blood types, such as the quantitative study of blood type antigens of the Kell, Rh and other systems.

[0005] Since flow cytometry requires that the sample to be tested be a single cell suspension, ABO blood type antigens that are easily agglutinated by antibodies cannot be directly detected by flow cytometry. Previously, in order to apply flow cytometry to the detection of ABO antigens, Berneman ZN et al. used dimethyl nitrite (DMS), formaldehyde or glutaraldehyde (GA) to treat red blood cells, and prepared FITC-IgG Fab fluorescent antibodies with low affinity to label the aldehyded red blood cells to evaluate the amount of AB antigens. They used this new method to detect a higher expression of AB antigens than the previous radioimmunoassay. However, their research also mentioned the defects of this method of fixing red blood cells: when the amount of glutaraldehyde is small, cell clots will increase; when the amount of glutaraldehyde is increased, the background value increases and the detection value decreases, resulting in increased errors. Red blood cells with higher antigen expression are prone to agglutination, and flow cytometry will exclude such agglutinated, high-expressing cells to ensure the accuracy of measuring the average value of single cells, resulting in measurement errors. Summary of the invention

[0006] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide an IgM antibody for preventing red blood cell agglutination and a preparation method and use thereof, so as to solve the problems in the prior art.

[0007] To achieve the above-mentioned object and other related objects, the present invention first provides an IgM antibody for preventing red blood cell agglutination, wherein the IgM antibody is obtained by mixing and incubating natural IgM antibody with DTT.

[0008] The present invention also provides a method for preparing the IgM antibody, which comprises the following steps: mixing a DTT solution with a natural IgM antibody and then incubating the mixture to obtain the IgM antibody for preventing red blood cell agglutination.

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

[0010] The present invention also provides a method for preparing the product, which comprises: 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 preparing a product for detecting ABO blood type and / or preparing a product for detecting the expression amount of type A antigen and / or type B antigen.

[0012] As described above, the IgM antibody for preventing red blood cell agglutination of the present invention and its preparation method and use have the following beneficial effects:

[0013] 1) The prepared IgM antibodies and / or products for preventing red blood cell agglutination can be used for direct quantitative detection of the expression of ABO blood type A antigen and / or B antigen in healthy people, detection of conventional ABO blood type reagent red blood cell products, study of the changes in the expression of different genotype ABO antigens under different physiological conditions, and study of diseases related to ABO antigen expression, etc.;

[0014] 2) Compared with the traditional method, the antibody / product for preventing red blood cell agglutination prepared in the present invention is simple to operate and has small batch differences when detecting the expression of type A antigen and / or type B antigen, and can be used for large-scale detection of ABO blood type and the expression of type A antigen and / or type B antigen. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic representation of the ability of the present invention to treat anti-A and anti-B blood group antigen antibodies with different concentrations of DTT to label ABO antigens Figure 1 .

[0016] Figure 2 Schematic representation of the ability of the present invention to treat anti-A and anti-B blood group antigen antibodies with different concentrations of DTT to label ABO antigens Figure 2 .

[0017] Figure 3 Schematic representation of the ability of the present invention to treat anti-A and anti-B blood group antigen antibodies with different concentrations of DTT to label ABO antigens Figure 3 .

[0018] Figure 4 The figure shows the flow cytometric analysis of the effect of anti-A blood type antigen antibody treated with 1 mM DTT for different days. (A) The figure shows the flow cytometric analysis of anti-A blood type antigen antibody treated with 1 mM DTT at 4°C / 7 days; (B) The figure shows the flow cytometric analysis of anti-A blood type antigen antibody treated with 1 mM DTT at 4°C / 3 days. DETAILED DESCRIPTION

[0019] The present invention firstly provides an IgM antibody for preventing red blood cell agglutination. The IgM antibody is obtained by mixing and incubating natural IgM antibody with DTT.

[0020] In certain embodiments of the present invention, the natural IgM antibody is an anti-type A and / or anti-type 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 reagent is a commercial reagent.

[0023] In the present invention, the concentration of DTT is determined by evaluating the ability of IgM antibodies to label antigens. The working concentration of DTT is obtained by the following steps: preparing DTT diluents of different dilution multiples, mixing and incubating each DTT diluent with natural IgM antibodies, and continuing to incubate the incubated system with type A antigen and / or type B antigen after the incubation, and determining the working concentration of DTT according to the concentration of the DTT diluent used in the group where no agglutination occurs after the incubation.

[0024] In certain embodiments of the present invention, the working concentration of DTT refers to the final concentration of the DTT diluent in the mixed solution after the natural IgM antibody and the DTT diluent are mixed, based on the volume of the mixed solution formed after the two are mixed.

[0025] In certain embodiments of the present invention, the absence of aggregation means that the agglutination result is shown as 1+w, ± or θ.

[0026] In certain embodiments of the present invention, the type A antigen and / or type B antigen may also be type A and / or type B red blood cells.

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

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

[0029] In a more preferred embodiment of the present invention, the ratio of the reciprocal of the natural IgM antibody titer to the DTT working concentration is 800: 0.5-1.5 mM. The ratio of the reciprocal of the natural IgM antibody titer to the DTT working concentration 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 most optimal embodiment of the present invention, the ratio of the reciprocal of the natural IgM antibody titer to the DTT working concentration is 800:1 mM.

[0031] In the present invention, the characteristics of red blood cells are not changed, only the reactivity of the antibody is changed, and the strong reducing property of DTT is used to avoid the antibody from causing red blood cell agglutination without affecting the specificity of the antibody. DTT can destroy the SS bond between IgM chains. In this method, DTT is added to natural IgM to process the 19S IgM multimer antibody into the 9S IgM monomer.

[0032] In the present invention, the IgM antibody can be used alone. If the IgM antibody is used alone, it needs to be prepared before use.

[0033] The present invention also provides a method for preparing the IgM antibody, which comprises the following steps: mixing a DTT solution with a natural IgM antibody and then incubating the mixture to obtain the IgM antibody for preventing red blood cell agglutination.

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

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

[0036] The working concentration of DTT is obtained by the following steps: preparing DTT diluents of different dilution multiples, mixing and incubating each DTT diluent with natural IgM antibody, and after the incubation, continuing to incubate the incubated system with type A antigen and / or type B antigen, and after the incubation, determining the working concentration of DTT according to the concentration of the DTT diluent used in the group where no agglutination occurs

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

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

[0039] In a more preferred embodiment of the present invention, the ratio of the reciprocal of the natural IgM antibody titer to the DTT working concentration is 800: 0.5-1.5 mM. The ratio of the reciprocal of the natural IgM antibody titer to the DTT working concentration 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 most optimal embodiment of the present invention, the ratio of the reciprocal of the natural IgM antibody titer to the DTT working concentration is 800:1 mM.

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

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

[0043] In certain 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 time is 7 days.

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

[0047] In the present invention, if the IgM antibody needs to be stored after preparation, it can be mixed with iodoacetamide to prepare the product for preventing red blood cell agglutination. Iodoacetamide is used to block free thiol groups. Since DTT has two thiol groups and can reduce two disulfide bonds, it is necessary to react with two iodoacetamide molecules. In the present invention, the amount of iodoacetamide in the product is determined by the amount of DTT added when preparing the IgM antibody.

[0048] In certain embodiments of the present invention, the iodoacetamide is in excess compared to DTT. The amount of DTT refers to the amount of DTT added when preparing the IgM antibody.

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

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

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

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

[0053] The present invention also provides a method for preparing the product, which comprises: 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 preparing a product for detecting ABO blood type and / or preparing a product for detecting the expression amount of type A antigen and / or type B antigen.

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

[0056] Before further describing the 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 terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0057] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

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

[0059] 1.1 Reagent preparation

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

[0061] 1.2 Antibody treatment

[0062] Mix 450 μL of DTT diluent and 450 μL of anti-A and anti-B blood typing reagent (the titer of -A / -B reagent is 1:800) in equal amounts. Mix thoroughly to obtain DTT concentrations of 1mM / 5mM / 10mM / 20mM in the mixed -A / -B reagent.

[0063] 1.3 Setting different reaction conditions

[0064] Divide 900 μL of DTT and -A / -B mixed solution into two EP tubes, mark them as 4°C and room temperature (25°C), respectively, and add 400 μL of DTT-treated -A / -B reagent into each EP tube.

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

[0066] At intervals of 1h / 2h / 4h / 24h / 48h / 72h, 50μL of DTT-treated-A / -B reagent was taken out from the EP tube and added to a small test tube, and then 50μL of type A / type B reagent red blood cells were added. After standing at room temperature for 15 minutes, centrifugation was performed to observe the results. The experimental results are shown in Table 1.

[0067] Table 1 Effect of different concentrations of DTT on the destruction of -A / -B cell agglutination at 4°C and room temperature

[0068]

[0069]

[0070] Note: 4+ represents a very strong agglutination reaction, and almost all cells have formed agglutination blocks; 3+ represents a strong agglutination reaction, and most cells have formed agglutination blocks; 2+ represents a moderate agglutination reaction, and some cells have formed agglutination blocks; 1+ represents a weak agglutination reaction, and only a small number of cells have formed agglutination blocks; ± represents extremely weak agglutination, which is manifested by the presence of a small number of cell agglutinations when the test tube is gently shaken, but the agglutination disappears when the test tube is continuously shaken; s represents "strong", indicating agglutination slightly stronger than the number marked, taking "1+s" as an example, "1+s" means agglutination slightly stronger than "1+", but less than the intensity of "2+" range; w represents "weak", indicating agglutination slightly weaker than the number marked, taking "1+w" as an example, "1+w" means agglutination slightly weaker than "1+", but the intensity is higher than the "±" range; θ represents no agglutination reaction, and the cells did not form agglutination.

[0071] According to Table 1, DTT concentrations higher than 1 mM, room temperature reaction for 24 h and DTT concentrations higher than 2 mM, 4°C reaction for 48 h can prevent -A / -B antibodies from agglutinating red blood cells. At 72 h, A / -B antibodies did not agglutinate red blood cells at all four DTT concentrations.

[0072] 1.5 Streaming Verification

[0073] Add 20 μL of -A / -B reagent treated with 1mM / 5mM / 10mM / 20mM DTT at room temperature / 4°C for 72 hours and 10 μL of type A / type B red blood cells into a small test tube and mix evenly, react at 4°C for 1 hour, wash three times with ice-cold PBS, add 1:500 diluted anti-mouse IgM-μ chain APC fluorescent secondary antibody, react at 4°C for 0.5 hour, wash three times with ice-cold PBS, resuspend to 800 μL, and detect by flow cytometry.

[0074] The flow cytometry results are shown in Table 2. In the table, the reaction conditions for the highest -A test value were DTT concentration 1mM, 4℃ treatment for 72h; the reaction conditions for the highest -B test value were DTT concentration 1mM, 25℃ treatment for 72h. The higher the -A and -B test values, the stronger the ability of the reagent to label the ABO antigen of red blood cells.

[0075] Table 2 Flow cytometry detection of labeling effects of -A / -B reagents treated with different concentrations of DTT 1

[0076]

[0077] 1.6 Preliminary Conclusions

[0078] According to Table 2 in 1.6, the lower the DTT concentration, the stronger the labeling ability of -A / -B reagent, and the effect of -A / -B reagent antibody treated at 4°C is better than that of antibody treated at room temperature. Among them, 4°C, 1mM DTT treatment of -A / -B reagent has the best effect. In order to further explore the most suitable DTT concentration and reaction conditions, this study continued to adjust the DTT concentration and extend the reaction time.

[0079] Example 2 Further exploration of the optimal reaction concentration and reaction time of DTT

[0080] 2.1 Reagent preparation and reaction conditions

[0081] As in 1.1-1.3, prepare 0.1mM / 0.25mM / 0.5mM / 1mM / 5mM / 10mM / 20mM DTT treatment-A / -B reagents respectively and react at room temperature and 4°C respectively.

[0082] 2.2 Effect of reagents on cell agglutination at different reaction times

[0083] At 24h / 72h / 168h (i.e. 1 day, 3 days, 7 days), take out 50μL of DTT-treated-A / -B reagent from the EP tube, add it to a small test tube, then add 50μL of type A / type B reagent red blood cells, place at room temperature for 15 minutes, and centrifuge to observe the results. 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 / 3 days are consistent with those in 1.4. When treated for 7 days, all reagents did not agglutinate red blood cells.

[0084] Table 3 Effect of different concentrations of DTT on the destruction of -A / -B cell agglutination at 4°C and room temperature

[0085]

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

[0087] 2.3 Stream Verification

[0088] According to Table 3, at room temperature, the -A / -B reagent was weakly agglutinated with 0.25mM / 0.5mM / 1mM DTT for 3 days. At 4°C, the -A / -B reagent was weakly agglutinated with DTT after 7 days of treatment with DTT concentration ≥1mM. The antibody's ability to agglutinate red blood cells was still weak when the DTT concentration was 0.1mM. In order to explore the most suitable DTT-treated antibody's optimal reaction concentration and reaction time, the -A / -B reagent antibody treated with DTT concentration of 0.25mM / 0.5mM / 1mM for 3 days at room temperature and the -A / -B reagent antibody treated with DTT concentration of 1mM / 5mM / 10mM / 20mM for 7 days at 4°C were selected. The method was the same as 1.5 to detect the effect of DTT-treated antibody labeling ABO antigen. The test results are shown in Table 4.

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

[0090]

[0091] Example 3 Determination of the Optimal DTT Treatment-A / -B Reagent Conditions

[0092] According to the streaming results in Table 2, we can draw Figure 1 The conditions were room temperature and 4°C, and incubated for 3 days. Combining the flow cytometry results in Table 2 and Table 4, the Figure 2 and Figure 3 .exist Figure 2 In the figure, -1 and -2 represent the results of 1.5 flow cytometry validation and 2.3 flow cytometry detection, respectively. Both 1.5 flow cytometry validation and 2.3 flow cytometry validation include the results of -A / -B antibody treated with 1 mM DTT for 3 days at room temperature, which are represented by 1 mM-1 and 1 mM-2.

[0093] in accordance with Figure 1 , Figure 2 At 4℃ and room temperature, the DTT concentration was 1mM, and the effect of DTT treatment on -A / -B antibody was the best. The effect of 4℃ treatment was better than that of room temperature, which may be because DTT was more gentle on the reduction of IgM in a 4℃ environment. Figure 2In the results, except for 1mM-1, the flow cytometry detection results of -A / -B were similar at the same DTT concentration, and the detection value of -A in 1mM-1 might be too high.

[0094] in accordance with Figure 3 When the DTT concentration is 1mM, the -A / -B reagents have a stronger ability to label ABO antigens after being treated at 4℃ for 3 days. However, after being treated at 4℃ for 7 days, the -A / -B reagents have a similar ability to label ABO antigens. Under this condition, the antigen labeling effect of the reagent may be more stable, and the flow cytometry graph ( Figure 4 ) shows that, taking -A as an example, the horizontal coordinates in Figures A and B are both forward scattered light (FSC), and the vertical coordinates are both side scattered light (SSC). The large cell group in the center of the figure is the target cell group for detection, which should be a single cell suspension. If there are cell clots in the sample to be tested, a small cell group will appear on the upper right side of the central cell group. There are significantly more agglutinated cells in Figure B than in Figure A. Therefore, when DTT is 1mM, the antibody-induced cell agglutination is less compared to the 4°C reaction for 7 days and 4°C reaction for 3 days. It is meaningless to continue to increase the number of reaction days because the continued increase in the number of days will cause DTT to inactivate.

[0095] Example 4 Determining the configuration method of the flow reagent for detecting highly expressed blood group antigens

[0096] The reaction conditions were selected to be suitable for the treatment of 7 days at 4°C with a DTT concentration of 1mM. That is, a dithiothreitol (DTT) solution was prepared with 0.1M, pH 7.4 PBS, and the DTT solution concentration was 2mM; the prepared 2mM DTT solution was fully mixed with the -A / -BIgM monoclonal antibody reagent and incubated at 4°C for 7 days. To ensure the stability of the reagent, an excess of iodoacetamide (IAA) was added to the -A / -B IgM reagent after incubation, so that the final concentration of the IAA solution was 5mM.

[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 Biotechnology; 0.1M, pH 7.2 PBS buffer: homemade in the laboratory, concentration; anti-A and anti-B blood typing reagents: batch number 20220711, Shanghai Blood Biology; -A direct labeled antibody: 9431PE, BGRL; -B direct labeled 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 amplifier: 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 Collection of ABO homozygous and heterozygous blood samples from healthy individuals

[0102] All AA homozygotes, AO heterozygotes, BB homozygotes, and BO heterozygotes were collected from blood samples of 555 healthy blood donors. AA / BB homozygotes and AO / BO heterozygotes were distinguished (using Chromas software) by amplifying and sequencing the E6 exon of type A and type B blood samples (sent to Sangon Biotech for sequencing). 20 homozygotes and heterozygotes of type A and type B were collected, totaling 80 test results, and the differences in the expression of A / B antigens were compared using the reagent antibody prepared by this patent and the traditional aldehyde method.

[0103] 5.3 The reagent prepared in this patent is used to detect the A / B antigen expression of pure / heterozygous ABO blood type in healthy people

[0104] Each blood sample was washed three times in 0.1 M PBS buffer and then prepared with 3% red blood cell suspension. TM Add 2.5 μL of red blood cell suspension to each well of the 96-well plate, and then add 5 μL of the reagent prepared in this patent to each well. After fully mixing, react at 4°C for 1 hour, wash 15 times with a curiox plate washer, then add a 1:500 diluted APC anti-mouse IgMμ chain fluorescent secondary antibody, mix thoroughly, incubate in the dark for 30 minutes, wash 15 times with a curiox plate washer, and transfer to a U-shaped 96-well plate with 200 μL PBS added to each well for flow detection. All samples were tested in each experiment, and the test was repeated 3 times independently. The negative control of the experiment was that no reagent prepared in this patent was added to each red blood cell suspension to be tested, only APC anti-mouse IgMμ chain fluorescent secondary antibody was added, and the other experimental conditions were the same, parallel experiments, and flow detection 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.

[0105] 5.4 Traditional hydroformylation method to detect the A / B antigen expression of ABO blood type homozygotes / heterozygotes in healthy people

[0106] Each blood sample was washed three times with PBS, and the supernatant was discarded to obtain the packed red blood cells. 10 μL of packed red blood cells were added to a small test tube, and then 330 μL of 0.01% glutaraldehyde solution (25% glutaraldehyde solution was diluted 1:250 in PBS) was added, and the mixture was shaken at room temperature for 10 minutes, and the supernatant was removed by centrifugation and washed three times to prepare a 3% red blood cell suspension. Due to the short hydroformylation time, 10 to 12 red blood cells were hydroformylated each time.

[0107] Take 2.5 μL of 3% red blood cell suspension from each tube and add the directly labeled PE fluorescent antibody for incubation. Add 10 μL of -A directly labeled antibody diluted 1:3.2 to each A-type red blood cell, and add 10 μL of -B directly labeled antibody diluted 1:4 to each B-type red blood cell. Add cells and PE directly labeled fluorescent antibody to Laminar Wash TM On a 96-well plate, mix evenly, incubate at room temperature in the dark for 30 minutes, wash 15 times with a curiox plate washer, transfer to a U-shaped 96-well plate with 200 μL PBS added to each well, and perform flow cytometry. All samples were tested in each experiment, and the experiment was repeated 3 times independently. The negative control of the experiment was that no directly labeled fluorescent secondary antibody was added to each aldehyded red blood cell suspension. The other experimental conditions were the same, and the experiments were conducted in parallel. 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.

[0108] 5.5 Comparison of the test results between this patented method and the traditional hydroformylation method

[0109] The results of three tests by the patented method and the traditional hydroformylation method are shown in Table 5. The mean values ​​were statistically tested, and a t-test was performed for paired samples of homozygotes and heterozygotes. When the p value is less than 0.05 or 0.01, there is a significant difference between homozygotes and heterozygotes; if it is significant, the mean values ​​(or differences) are specifically compared to describe the specific differences. As shown in Table 5, in the test results, 8 groups of paired data showed differences (p<0.05), including 2 groups of type B (groups 1 and 3), 2 groups of type A (groups 2 and 3) in the hydroformylation method, and 1 group of type B (group 3) and 3 groups of type A (groups 1, 2 and 3) in the new method. In the hydroformylation method, the t values ​​of the three groups of type B are >0 for 2 groups and <0 for one group, indicating that the test values ​​of group 2 indicate that the expression of BB homozygotes is higher than that of BO heterozygotes, and the test values ​​of group 1 indicate that the expression of BB homozygotes is lower than that of BO heterozygotes. The t values ​​of the three groups of type A by the hydroformylation method were all <0, indicating that the expression level of AA homozygotes was lower than that of AO heterozygotes. In the new method, the t values ​​of all the test groups of type A and type B were all >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 Detection values ​​of ABO antigen expression in healthy people by flow cytometry using the new method of this patent and the hydroformylation method

[0111]

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

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

[0114] The p value less than 0.05 or 0.01 indicates that there are significant differences among the three groups. It can be seen that in the hydroformylation method, only the detection values ​​of the three groups of BB, that is, type B homozygotes, have no significant differences and small differences between batches, while the differences between batches of BO / AA / AO are large. The p values ​​of the new method are all greater than 0.01, indicating that the detection results of the new method have small differences between batches.

[0115] The F value is a statistic that measures the ratio of the difference between groups to the difference within groups. The larger the F value, the greater the difference between groups relative to the difference within groups, which means that the difference between different groups is more significant. The F values ​​of the three groups of the hydroformylation methods BO / AA / AO are much larger than those of the new method. The F values ​​of the new method test results are relatively stable.

[0116] Table 6 Results of variance analysis

[0117]

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

[0119] 5.6 Evaluation of test results

[0120] According to the test results, the new method has small batch differences in the test results, and the test results of the new method are considered more reliable. It can be concluded that the A / B antigen expression of type A and type B homozygotes is higher than that of heterozygotes, indicating that the expression of normal ABO blood type antigen is related to the ABO gene. Both the traditional method and the new detection system can detect the ABO antigen expression of ABO homozygotes and heterozygotes, but the experimental process of the aldehyde red blood cell method is complicated and time-consuming, and the test results of different batches of red blood cells vary greatly, and the results are unstable. The experimental process of the newly established IgM antibody detection system is simple, and there is no obvious difference between the groups.

[0121] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and the variations of the methods in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present 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 the present invention.

Claims

1. An IgM antibody for preventing hematocrit agglutination, characterized in that: The IgM antibody is obtained by mixing and incubating natural IgM antibody with DTT.

2. The IgM antibody according to claim 1, characterized in that The natural IgM antibody is an anti-A type antigen antibody and / or an anti-B type antigen antibody.

3. The IgM antibody according to claim 1, characterized in that The working concentration of DTT is obtained by the following steps: preparing DTT diluents with different dilution multiples, mixing and incubating each DTT dilution with natural IgM antibody, and continuing to incubate the incubated system with type A antigen and / or type B antigen after the incubation, and determining the working concentration of DTT according to the concentration of the DTT dilution used in the group where no agglutination occurs after the incubation.

4. The IgM antibody according to claim 1, characterized in that The ratio of the reciprocal of the natural IgM antibody titer to the DTT working concentration is 800:0.25-5mM; preferably, the ratio of the reciprocal of the natural IgM antibody titer to the DTT working concentration is 800:0.5-2mM.

5. The method for preparing an IgM antibody according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: mixing a DTT solution with a natural IgM antibody and then incubating the mixture to obtain the IgM antibody for preventing red blood cell agglutination.

6. The method for preparing an IgM antibody according to claim 5, characterized in that: Includes any one or more of the following conditions: 1) The working concentration of the DTT solution is obtained by the following steps: preparing DTT diluents with different dilution multiples, mixing and incubating each DTT diluent with natural IgM antibody, and after the incubation, continuing to incubate the incubated system with type A antigen and / or type B antigen, and after the incubation, determining the working concentration of DTT according to the concentration of the DTT diluent used in the group where no agglutination occurs; 2) the DTT solution is obtained by mixing DTT and a buffer solution; 3) The ratio of the reciprocal of the natural IgM antibody titer to the DTT working concentration is 800: 0.25-5 mM; preferably, the ratio of the reciprocal of the natural IgM antibody titer to the DTT working concentration is 800: 0.5-2 mM; 4) The incubation temperature is 2-6°C; 5) The incubation time is 24 hours to 7 days; preferably, the incubation time is 3 to 7 days.

7. A product for preventing red blood cell agglutination, characterized in that: The product comprises the IgM antibody according to any one of claims 1 to 4 and iodoacetamide.

8. The product according to claim 7, characterized in that The iodoacetamide is in excess compared to DTT; preferably, the molar ratio of the iodoacetamide to DTT is 1:2-3.

9. The method for preparing the product according to any one of claims 7 to 8, characterized in that: The preparation method comprises mixing iodoacetamide with the IgM antibody according to any one of claims 1 to 4 to obtain the product.

10. Use of the IgM antibody according to any one of claims 1 to 4 or the product according to any one of claims 7 to 8 in preparing a product for detecting ABO blood type and / or preparing a product for detecting the expression of type A antigen and / or type B antigen.

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