Erythrocyte adhesion enhancer as well as preparation method and application thereof

Abstract

The invention discloses a red blood cell attachment enhancer, and every 1000 mL of the red blood cell attachment enhancer comprises the following components: 0.5 to 2.0 g of sodium chloride, 2.0 to 5.0 g of disodium ethylene diamine tetraacetate, 1.0 to 3.0 g of sodium citrate, and the balance of ultrapure water. The erythrocyte attachment enhancer and the washed human packed erythrocytes form a cell suspension which can be used for preparing a solid-phase agglutination reaction microplate. Compared with the traditional'normal saline-red blood cell 'suspension, the cell suspension constructed by the red blood cell attachment enhancer and the human packed red blood cells can obviously increase the number of red blood cells attached to the surface of a solid-phase microplate carrier, ensures sufficient and stable number of red blood cell antigens attached to the solid-phase microplate in the long-term preservation process, and has the advantages of simple operation and high efficiency. The number of red blood cell antigens reacting with the antibody to be detected is ensured, the sensitivity of a solid-phase agglutination reaction is improved, false negative caused by leak detection of the same antibody is avoided, and the blood transfusion risk is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of diagnostic reagent preparation, and specifically, to a reagent preparation for enhancing the adhesion of red blood cells to the surface of a solid-phase carrier and related applications. Background Art

[0002] The antigen-antibody reaction generally includes two stages: the first stage is the random collision and mutual binding of antigens and antibodies in the reaction system. This stage occurs extremely fast and is affected by various factors, usually not visible; the second stage is the complete binding of antigens and antibodies. This stage requires a long time and needs to be enhanced during the experiment to achieve visualization. Common serological antigen-antibody reactions include: agglutination reaction, sensitization reaction, hemolysis reaction, neutralization reaction, and precipitation reaction. The factors affecting these reactions are: proteolytic enzymes, high molecular polymers, low ionic strength salt solutions, the pH of the reaction system, the electric potential energy of the reaction system, the number of antigens and antibodies, etc.

[0003] Red blood cells play the role of antigens in the serological antigen-antibody reaction and can detect antibodies in serum. Currently, the main methods for detecting antibodies in serum are the microcolumn gel card method and the solid-phase agglutination method. The solid-phase agglutination method has a large detection throughput and a relatively long shelf life of the reagent kit, and both have good applications in clinical practice. When detecting antibodies in serum by either the gel card method or the solid-phase agglutination method, it is first necessary to ensure that a sufficient number of red blood cell antigens react with the antibodies in the serum to avoid false negatives and missed detection of antibodies. In current actual work, when preparing a solid-phase agglutination reaction microplate conventionally, red blood cells are mainly suspended in physiological saline or other buffer solutions and then added to the pre-coated microplate wells, so as to adhere to the bottom of the microplate wells, and then react with the antibodies in the serum to be tested, ultimately achieving the purpose of detecting antibodies. Conventional physiological saline or buffer solutions are mainly electrically neutral. After suspending red blood cells with them, the charge on the surface of red blood cells cannot be adjusted and stabilized, and it can only rely on the natural sedimentation of red blood cells to bind to the positive charge of the microplate wells for adhesion. At the same time, under the action of gravity, the conventionally suspended red blood cells quickly sediment into clusters and cannot form a uniform monolayer of red blood cells, without any promotion or enhancement effect on the adhesion of red blood cells. Therefore, the adhesion efficiency of red blood cells suspended in physiological saline or buffer solutions is low. When the number of adhered red blood cells is insufficient, it means that the number of red blood cell antigens is relatively insufficient, and there are not enough red blood cell antigens to bind to the serum antibodies per unit time. For antibodies with a dose effect in the serum to be tested, it may lead to missed detection and false negative results, which are likely to cause blood transfusion risks and need to be solved. Summary of the Invention

[0004] Aiming at the problems existing in the existing detection methods, the purpose of the present invention is to provide a red blood cell adhesion enhancer and its application in the preparation of a solid-phase agglutination reaction microplate, which can ensure the number of red blood cells participating in the reaction as antigen substances, avoid the occurrence of false negative results in the detection method, and ensure the accuracy of the detection results.

[0005] The red blood cell adhesion enhancer provided by the present invention is characterized in that each 1000 mL of the red blood cell adhesion enhancer contains the following components in mass: 0.5 - 2.0 g of sodium chloride, 2.0 - 5.0 g of disodium ethylenediaminetetraacetate, 1.0 - 3.0 g of sodium citrate, and the balance is ultrapure water.

[0006] Preferably, each 1000 mL of the red blood cell adhesion enhancer contains the following components: 1.0 g of sodium chloride, 4.5 g of disodium ethylenediaminetetraacetate, 1.5 g of sodium citrate, and the balance is ultrapure water.

[0007] Preferably, each 1000 mL of the red blood cell adhesion enhancer contains the following components: 1.0 g of sodium chloride, 3.0 g of disodium ethylenediaminetetraacetate, 2.0 g of sodium citrate, and the balance is ultrapure water.

[0008] Preferably, each 1000 mL of the red blood cell adhesion enhancer contains the following components: 1.0 g of sodium chloride, 5.0 g of disodium ethylenediaminetetraacetate, 2.0 g of sodium citrate, and the balance is ultrapure water.

[0009] The present invention provides a preparation method of the red blood cell adhesion enhancer. The steps include: stirring each component in ultrapure water, uniformly dissolving it, and then filtering and sterilizing it with a 0.22 μm filter.

[0010] The present invention also provides the application of the red blood cell adhesion enhancer in the preparation of a solid-phase agglutination reaction microplate for detecting red blood cell antibodies.

[0011] Specifically, the preparation process of the solid-phase agglutination reaction microplate is as follows: preparing a cell suspension with a certain concentration using the red blood cell adhesion enhancer and human red blood cells, dispensing the cell suspension into a 96-well microplate that has been coated using a multi-channel pipette, 100 μL per well, standing in a 4°C refrigerator for at least 7 hours, washing, lysing with 1×PBS, and then adding a drying protective solution and drying to obtain it.

[0012] Preferably, the cell suspension is prepared by mixing the red blood cell adhesion enhancer and human packed red blood cells at a volume ratio of 99.4∶0.6.

[0013] Compared with the traditional method of suspending red blood cells with physiological saline, the red blood cell adhesion enhancer and its application of the present invention have the following advantages and beneficial effects: 1. In the red blood cell adhesion enhancer, disodium ethylenediaminetetraacetate (EDTA-Na 2) and sodium citrate mainly chelate divalent cations such as cellular Ca 2+ , Mg 2+ etc., regulate and stabilize the charge distribution on the cell membrane surface, so that red blood cells do not undergo rapid sedimentation and aggregation, but adhere to the positive charge on the surface of the solid-phase carrier in the form of a single cell layer and attach, and are evenly distributed on the surface of the solid-phase carrier, facilitating subsequent lysis of the red blood cell monolayer.

[0014] 2. While low-concentration sodium chloride is mainly used to adjust the osmotic pressure, provide a stable reaction system environment, and cooperate with disodium ethylenediaminetetraacetate and sodium citrate to enhance the firm adhesion of red blood cells in the microplate coated with a cationic charge matrix.

[0015] 3. Using the red blood cell adhesion enhancer of the present invention can stabilize the charge on the cell membrane surface, which is beneficial to reducing damage to the cell membrane, so that the blood shadow cell membrane antigen obtained after lysis of the red blood cell monolayer maintains the greatest degree of integrity.

[0016] After suspending red blood cells with the red blood cell adhesion enhancer of the present invention, the number of red blood cells adhering in the microplate wells can be increased, the adhesion efficiency is improved, so that when detecting plasma antibodies, there are sufficient red blood cell antigens to react with antibodies per unit time, and finally the purpose of detecting dose-effect antibodies is achieved. It avoids the false negative problem of missed detection of antibodies due to insufficient adhesion of membrane antigens in the traditional "saline-red blood cell" suspension plate making, and improves the detection quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is for observing the distribution state of blood shadow cells after lysis of red blood cells under a microscope; Figure 1 A- Figure 1 D respectively correspond to the distribution states of blood shadow cells after lysis of red blood cells in the 1st to 4th solid-phase detection microplates.

[0018] Figure 2 It is the result of testing anti-Fya on the solid-phase detection microplates prepared by suspending red blood cells with different formulations of adhesion enhancers and suspending red blood cells with physiological saline; Figure 2 A- Figure 2 D respectively correspond to the distribution states of blood shadow cells after lysis of red blood cells in the 1st to 4th solid-phase detection microplates.

[0019] Figure 3 It is the test result of the test plate and the control plate on dose-effect antibodies (anti-JKb and anti-Fya); Figure 3 A, Figure 3 B respectively correspond to the test results of the test plate and the control plate.

[0020] Figure 4 It is the test result of the stability of the test plate and the control plate; Figure 4 A, Figure 4B corresponds to the test results of the test plate and the control plate respectively. Detailed implementation mode

[0021] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will also become clearer with the description. However, these embodiments are exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that without departing from the spirit and scope of the present invention, modifications or substitutions can be made to the details and forms of the present invention, but these modifications and substitutions all fall within the protection scope of the present invention.

[0022] In the following examples, all raw materials are purchased from reagent companies, generally experimental reagents with a purity of ≥98%. Example 1

[0023] A preparation method of a red blood cell adhesion enhancer includes the following steps: Weigh 1.0 g of sodium chloride, 4.5 g of disodium ethylenediaminetetraacetate, and 1.5 g of sodium citrate, and the balance is made up to 1000 mL with purified water. After being prepared according to the formula ratio, it is stirred evenly by a magnetic stirrer with heating, and then vacuum filtered and sterilized with a filter with a pore size of 0.22 μm to obtain the red blood cell adhesion enhancer.

[0024] Based on Example 1, Examples 2-3 are set. In Examples 2-3, except for the different weights of disodium ethylenediaminetetraacetate and sodium citrate, other processes remain unchanged. The specific component contents corresponding to the 3 examples are shown in Table 1 below: Table 1 Component contents in Examples 1-3

[0025] After suspending red blood cells with the red blood cell adhesion enhancers prepared in Examples 1-3 and normal saline respectively, a solid-phase agglutination reaction microplate is prepared. The specific preparation steps are as follows.

[0026] 1. Preparation of cell suspension The red blood cell adhesion enhancers obtained in Examples 1-3 and the packed red blood cells after washing to remove plasma and buffy coat are respectively prepared into cell suspensions according to a volume ratio of 99.4:0.6. After mixing evenly, a red blood cell adhesion enhancer-red blood cell suspension is obtained.

[0027] A normal saline-red blood cell suspension is obtained by mixing normal saline and the packed red blood cells after washing to remove plasma and buffy coat, as a control example.

[0028] 2. Preparation of solid-phase reaction microplate The 4 types of cell suspensions prepared in Step 1 were respectively aliquoted into a 96-well U-shaped microplate that had been coated, with 100 μL per well, and then placed in a refrigerator at 4°C for at least 7 hours. After taking out, it was washed several times with physiological saline. After lysing red blood cells with 1×PBS, it was washed again, and then a cryoprotectant was added to prepare a dry-type microplate for solid-phase detection. Among them, the microplates for solid-phase detection prepared with the red blood cell adhesion enhancers of Application Examples 1-3 were respectively labeled as Microplate for Solid-Phase Detection No. 1, Microplate for Solid-Phase Detection No. 2, and Microplate for Solid-Phase Detection No. 3; the microplate for solid-phase detection prepared with physiological saline was labeled as Microplate for Solid-Phase Detection No. 4.

[0029] Experiments were conducted on Microplates for Solid-Phase Detection No. 1-4.

[0030] Experiment 1: Observe under a microscope whether the distribution of ghost cells after red blood cell lysis in Microplates for Solid-Phase Detection No. 1-4 is uniform and whether the quantity is sufficient. The specific results are as follows: In Microplate for Solid-Phase Detection No. 1, the number of ghost cells formed after red blood cell lysis was relatively large, evenly and densely distributed in the center and at the edge of the well, without eccentric or lateral distribution with missing corners or blanks, which was relatively ideal ( Figure 1 A); In Microplate for Solid-Phase Detection No. 2, the distribution of ghost cells formed after red blood cell lysis was relatively uniform, with both in the center and at the edge of the well, but the density was still on the low side ( Figure 1 B); In Microplate for Solid-Phase Detection No. 3, the number of ghost cells formed after red blood cell lysis was acceptable, but the distribution was uneven, with a lateral distribution phenomenon ( Figure 1 C); In Microplate for Solid-Phase Detection No. 4, the number of ghost cells formed after red blood cell lysis was small, and the distribution was acceptable ( Figure 1 D).

[0031] Experiment 2: The anti-Fya plasma with an agglutination strength of 2+ determined by the microcolumn gel card method was tested using Microplates for Solid-Phase Detection No. 1-4, and repeated 3 times. The test results are as follows: Microplate for Solid-Phase Detection No. 2 could detect anti-Fya, which was equivalent to the microcolumn gel card method ( Figure 2 B); Microplate for Solid-Phase Detection No. 3 could detect anti-Fya, slightly stronger than the microcolumn gel card method, but there was a missing corner phenomenon ( Figure 2 C); Microplate for Solid-Phase Detection No. 1 could detect anti-Fya, relatively strongly, stronger than the microcolumn gel card method ( Figure 2 A); Microplate for Solid-Phase Detection No. 4 could detect anti-Fya, but weaker than the microcolumn gel card method ( Figure 2 D); According to the results of Experiment 1 and Experiment 2, it can be concluded that the effect of suspending red blood cells in Microplates for solid-phase detection No. 1-3 is stronger than that in Microplate for solid-phase detection No. 4, and the effect of Microplate for solid-phase detection No. 1 is the best. That is, when the red blood cell adhesion enhancer disclosed in the present invention suspends red blood cells, it can promote the adhesion of red blood cells on the microplate wells compared with suspension with physiological saline.

[0032] Next, the performance of the two was comprehensively evaluated using Microplate for solid-phase detection No. 1 (test plate) and Microplate for solid-phase detection No. 4 (control plate).

[0033] 1. Test of dose-effect antibodies on the test plate and the control plate The antibodies with dose effect in the Kidd blood group system (anti-JKb) and Duffy blood group system (anti-Fya) were titer tested by the microcolumn gel card method to determine the titers. Subsequently, the prepared solid-phase agglutination test plate and control plate were used for titer testing. By comparison, the difference in reaction sensitivity between the reaction plates prepared by suspending red blood cells with the red blood cell adhesion enhancer and physiological saline was obtained.

[0034] The test results showed that the titers of anti-JKb and anti-Fya measured by the microcolumn gel card method were 8 and 64 respectively, and the titers measured by the test plate were 128 and 128 respectively, both higher than those measured by the microcolumn gel card method ( Figure 3 A); the titers measured by the control plate were 64 and 32 respectively( Figure 3 B), both lower than the titers measured by the test plate. This indicates that the test plate is superior to the control plate and the microcolumn gel card method in detecting dose-effect antibodies.

[0035] 2. Stability test of the test plate and the control plate Three batches of solid-phase agglutination reaction test plates prepared by suspending red blood cells with the red blood cell adhesion enhancer and three batches of control plates prepared by suspending red blood cells with physiological saline were used synchronously. Anti-JKb and anti-Fya with a card reading of 2+ were tested at 1 month, 3 months, 5 months, and 7 months of storage, focusing on whether there were changes in the agglutination intensity and comparing the reaction intensity differences between the test plate and the control plate during each storage period.

[0036] The test results showed that: during the 7-month observation period of the three batches of solid-phase agglutination reaction test plates prepared by suspending red blood cells with the red blood cell adhesion enhancer, the agglutination intensity remained stable, with a difference of no more than 1+, and the negative and positive controls were distinct ( Figure 4 A); during the 7-month observation period of the three batches of control plates prepared by suspending red blood cells with physiological saline, the stability of the agglutination intensity was poor, with a difference reaching 2+, and the negative and positive controls were distinct ( Figure 4B). Overall comparison shows that the agglutination intensity of the solid-phase agglutination reaction test plates prepared with red blood cell adhesion enhancer-suspended red blood cells is stronger than that of the control plates prepared with normal saline-suspended red blood cells during the same period. When stored for 5 months and 7 months, the difference in agglutination intensity between the test plates and the control plates for the detection of related antibodies is about 2+, indicating the good advantage of the solid-phase microplates prepared with red blood cell adhesion enhancer in detecting dose-effect antibodies.

[0037] 3. Precision testing of test plates and control plates Three batches of solid-phase agglutination reaction test plates prepared with red blood cell adhesion enhancer-suspended red blood cells were used to test 94 samples (47 positive and 47 negative) at different times, in different laboratories, and by different operators to compare whether the results of the three batches of solid-phase reaction test plates were consistent to evaluate precision.

[0038] The test results show that when tested in different laboratories and by different operators, the results measured by the test plates are completely consistent, without false positives and false negatives, indicating that the precision of the test plates is good; among the control plates in the synchronous parallel tests, there are differences in the test results between 7 months of storage and 1 month and 3 months, and the agglutination intensity of positive specimens generally decreases by one agglutination intensity, indicating that the precision of the control plates is poor.

[0039] 4. Testing of serum specimens with test plates and control plates (1) Ninety-four positive serum specimens containing irregular antibodies whose specificity was determined by the microcolumn gel card method were tested using the test plates and control plates respectively, and the results were compared with those of the microcolumn gel card method; (2) Ninety-four normal negative sera confirmed by the microcolumn gel card method not containing irregular antibodies were tested using the test plates and control plates respectively, and the results were compared with those of the microcolumn gel card method.

[0040] The test results of the test plates and control plates for positive and negative specimens are shown in Table 2. Among the specimens detected as positive by the test plates, 8 cases showed negative results in the control plates. The chi-square statistical results indicate that the difference between the two is statistically significant (X 2 = 8, P < 0.005), indicating that the serum test results of the test plates are better than those of the control plates.

[0041] Table 2 Test results of test plates and control plates for serum specimens .

Claims

1. A red blood cell adhesion enhancer, characterized in that: Each 1000 mL of the red blood cell adhesion enhancer contains the following components: 0.5-2.0 g of sodium chloride, 2.0-5.0 g of disodium ethylenediaminetetraacetate, 1.0-3.0 g of sodium citrate, and the balance is ultrapure water.

2. The red blood cell adhesion enhancer according to claim 1, characterized in that Each 1000 mL of red blood cell attachment enhancer contains the following components: 1.0 g of sodium chloride, 4.5 g of disodium ethylenediaminetetraacetate, 1.5 g of sodium citrate, and the balance is ultrapure water.

3. The red blood cell adhesion enhancer according to claim 1, characterized in that Each 1000 mL of the red blood cell adhesion enhancer contains the following components: 1.0 g of sodium chloride, 3.0 g of disodium ethylenediaminetetraacetate, 2.0 g of sodium citrate, and the balance is ultrapure water.

4. The red blood cell adhesion enhancer according to claim 1, characterized in that Each 1000 mL of the red blood cell adhesion enhancer contains the following components: 1.0 g of sodium chloride, 5.0 g of disodium ethylenediaminetetraacetate, 2.0 g of sodium citrate, and the balance is ultrapure water.

5. A method for preparing the red blood cell adhesion enhancer according to any one of claims 1 to 4, characterized in that: The steps are as follows: sodium chloride, disodium ethylenediaminetetraacetate and sodium citrate are added into ultrapure water and stirred, and after being uniformly dissolved, the mixture is filtered and sterilized with a 0.22 um filter.

6. Use of the red blood cell adhesion enhancer according to any one of claims 1 to 4 in preparing a solid phase agglutination reaction microplate for red blood cell antibody detection.

7. The use according to claim 6, characterized in that In the process of preparing the solid phase agglutination reaction microplate, the red blood cell adhesion enhancer and human red blood cells are first used to prepare a cell suspension, and then the cell suspension is separated into the coated microplate wells by a spray gun.

8. The use according to claim 7, characterized in that The cell suspension is prepared by a red blood cell adhesion enhancer and human packed red blood cells in a volume ratio of 99.4:0.6.

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