A red blood cell adhesion enhancer, its preparation method and application

Abstract

The application discloses a red blood cell adhesion enhancer, wherein 1000 mL of the red blood cell adhesion enhancer comprises 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 rest is ultrapure water. The red blood cell adhesion enhancer and the cell suspension of washed human packed red blood cells can be used for preparing a solid-phase agglutination reaction microplate. Compared with a traditional "normal saline-red blood cell" suspension, the cell suspension constructed by the red blood cell adhesion enhancer and the human packed red blood cells can significantly increase the number of red blood cells adhered to the surface of a solid-phase microplate carrier, ensure the sufficient and stable number of adhered red blood cell antigens in a long-term storage process of the solid-phase microplate, ensure the number of red blood cell antigens reacted with a to-be-tested antibody, improve the sensitivity of the solid-phase agglutination reaction, avoid false negatives caused by missed detection of the same antibody, and reduce the risk of blood transfusion.

Description

Technical Field

[0001] This invention relates to the field of diagnostic reagent preparation technology, and more specifically, to the preparation and related applications of a reagent for enhancing the adhesion of erythrocytes to the surface of a solid carrier. Background Technology

[0002] Antigen-antibody reactions typically involve two phases: the first phase involves random collisions and binding of antigens and antibodies within the reaction system; this phase occurs extremely rapidly and is influenced by various factors, usually remaining invisible. The second phase involves complete antigen-antibody binding, which requires a longer time and necessitates enhancement during the experiment to achieve visualization. Common serological antigen-antibody reactions include agglutination, sensitization, hemolysis, neutralization, and precipitation. Factors affecting these reactions include proteolytic enzymes, high molecular weight polymers, low ionic strength salt solutions, the pH of the reaction system, the electrical potential of the reaction system, and the quantity of antigens and antibodies.

[0003] Red blood cells act as antigens in serological antigen-antibody reactions, enabling the detection of antibodies in serum. Currently, the main methods for detecting antibodies in serum are microcolumn gel card assays and solid-phase agglutination assays. Solid-phase agglutination assays, due to their high throughput and long shelf life, are widely used in clinical practice. Whether using gel card assays or solid-phase agglutination assays to detect antibodies in serum, it is crucial to ensure a sufficient quantity of red blood cell antigens react with serum antibodies to avoid false negatives and missed antibody detection. In practice, the routine preparation of solid-phase agglutination reaction microplates involves suspending red blood cells in physiological saline or other buffer solutions, adding them to the pre-coated wells of the microplate, allowing them to adhere to the bottom of the wells, and then reacting with antibodies from the serum to be tested, ultimately achieving antibody detection. However, conventional physiological saline or buffer solutions are primarily electrically neutral; suspending red blood cells in these solutions cannot regulate or stabilize the surface charge of the red blood cells, relying solely on the natural sedimentation of the red blood cells to bind with the positively charged microplate wells for adhesion. Meanwhile, conventionally suspended red blood cells quickly settle into clumps under gravity, failing to form a uniform monolayer. This provides no promotion or enhancement to red blood cell adhesion, resulting in low adhesion efficiency for red blood cells suspended in physiological saline or buffer solutions. When the number of adhered red blood cells is insufficient, it means a relative insufficiency in red blood cell antigens. Insufficient red blood cell antigens bind to serum antibodies per unit time, potentially leading to false negatives in serum with dose-dependent antibodies, posing a significant risk of transfusion complications. This issue needs to be addressed. Summary of the Invention

[0004] To address the problems existing in current detection methods, the purpose of this invention is to provide a erythrocyte adhesion enhancer and its application in the fabrication of solid-phase agglutination reaction microplates. This enhancer ensures the quantity of erythrocytes participating in the reaction as antigens, avoids false negative results in detection methods, and ensures the accuracy of detection results.

[0005] The erythrocyte adhesion enhancer provided by the present invention is characterized in that each 1000 mL of the erythrocyte adhesion enhancer contains the following components by 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 being ultrapure water.

[0006] Preferably, each 1000 mL of erythrocyte adhesion enhancer contains the following components: 1.0 g sodium chloride, 4.5 g disodium EDTA, 1.5 g sodium citrate, and the remainder is ultrapure water.

[0007] Preferably, each 1000 mL of the erythrocyte adhesion enhancer comprises the following components: 1.0 g sodium chloride, 3.0 g disodium EDTA, 2.0 g sodium citrate, and the remainder being ultrapure water.

[0008] Preferably, each 1000 mL of the erythrocyte adhesion enhancer comprises the following components: 1.0 g sodium chloride, 5.0 g disodium EDTA, 2.0 g sodium citrate, and the balance being ultrapure water.

[0009] This invention provides a method for preparing a red blood cell adhesion enhancer, the steps of which include: stirring each component in ultrapure water, dissolving it evenly, and then filtering it through a 0.22µm filter to remove bacteria.

[0010] This invention also provides the application of erythrocyte adhesion enhancers in the preparation of solid-phase agglutination reaction microplates for erythrocyte antibody detection.

[0011] Specifically, the solid-phase aggregation reaction microplate preparation process is as follows: A cell suspension of a certain concentration is prepared using erythrocyte adhesion enhancer and human erythrocytes. The cell suspension is then separated into 96-well microplates that have been coated using a pipette, 100 μL / well. The plates are incubated at 4°C for at least 7 hours, washed, lysed with 1×PBS, and then dried with desiccant to obtain the final product.

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

[0013] Compared to the traditional method of suspending red blood cells in physiological saline, the red blood cell adhesion enhancer of the present invention and its application have the following advantages and beneficial effects:

[0014] 1. Among erythrocyte adhesion enhancers, disodium ethylenediaminetetraacetate (EDTA-Na2) and sodium citrate mainly work by chelating cell calcium. 2+ Mg 2+ The presence of divalent cations regulates and stabilizes the charge distribution on the cell membrane surface, preventing red blood cells from rapidly settling and clumping together. Instead, they adhere to the solid carrier surface as a monolayer by binding with the positive charge and are evenly distributed on the solid carrier surface, which facilitates subsequent red blood cell monolayer lysis.

[0015] 2. Low concentrations of sodium chloride are mainly used to regulate osmotic pressure and provide a stable reaction environment. It works synergistically with disodium ethylenediaminetetraacetate and sodium citrate to enhance the firm adhesion of red blood cells in microplates coated with cationic charge matrix.

[0016] 3. The erythrocyte adhesion enhancer of the present invention can stabilize the surface charge of the cell membrane, which helps to reduce cell membrane damage and ensures that the blood shadow cell membrane antigen obtained after the lysis of the erythrocyte monolayer maintains the maximum integrity.

[0017] By suspending erythrocytes using the erythrocyte adhesion enhancer of this invention, the number of erythrocytes adhering to the wells of the microplate can be increased, improving adhesion efficiency. This ensures that sufficient erythrocyte antigens react with antibodies per unit time when detecting plasma antibodies, ultimately achieving the goal of detecting dose-effect antibodies. This avoids the false negative problem of missed antibody detection due to insufficient membrane antigen adhesion in traditional "physiological saline-erythrocyte" suspension plate preparation, thus improving detection quality. Attached Figure Description

[0018] Figure 1 To observe the distribution of shadow cells after erythrocyte lysis under a microscope; Figure 1 A- Figure 1 D corresponds to the distribution of shadow cells after erythrocyte lysis in microplates 1-4 for solid-phase detection.

[0019] Figure 2 The results of anti-Fya were tested on solid-phase microplates prepared by attaching different formulations of red blood cells with enhancers and red blood cells suspended in physiological saline. Figure 2 A- Figure 2 D corresponds to the distribution of shadow cells after erythrocyte lysis in microplates 1-4 for solid-phase detection.

[0020] Figure 3 The test results for dose-effect antibodies (anti-JKb and anti-Fya) are shown for the experimental and control plates. Figure 3 A, Figure 3 B corresponds to the test results of the experimental panel and the control panel, respectively.

[0021] Figure 4 The results show the stability test results for the experimental and control panels. Figure 4 A, Figure 4 B corresponds to the test results of the experimental panel and the control panel, respectively. Detailed Implementation

[0022] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the present invention can be made without departing from the spirit and scope of the invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0023] In the following examples, all raw materials were purchased from reagent companies and are generally laboratory reagents with a purity of ≥98%. Example 1

[0024] A method for preparing a red blood cell adhesion enhancer includes the following steps: weighing 1.0g of sodium chloride, 4.5g of disodium ethylenediaminetetraacetate, and 1.5g of sodium citrate, with the remainder being purified water to a final volume of 1000mL, preparing the mixture according to the formula ratio, heating and stirring evenly with a magnetic stir bar, and then vacuum filtering and sterilizing using a filter with a pore size of 0.22μm to obtain the red blood cell adhesion enhancer.

[0025] Based on Example 1, Examples 2 and 3 were set up. The only difference between Examples 2 and 3 was the weight of disodium ethylenediaminetetraacetate and sodium citrate; all other processes remained unchanged. The specific component contents corresponding to the three examples are shown in Table 1 below:

[0026] Table 1 Content of each component in Examples 1-3

[0027]

[0028] After suspending red blood cells in the red blood cell adhesion enhancer prepared in Examples 1-3 above and physiological saline respectively, solid-phase agglutination reaction microplates were prepared. The specific preparation steps are as follows.

[0029] 1. Preparation of cell suspension

[0030] The erythrocyte adhesion enhancer obtained in Examples 1-3 and the hematocrit erythrocytes after washing and removing plasma and white membrane layers were respectively prepared into cell suspensions at a volume ratio of 99.4:0.6. After being mixed evenly, the erythrocyte adhesion enhancer-erythrocyte suspension was obtained.

[0031] A saline-red blood cell suspension was prepared by mixing physiological saline with washed red blood cells after removing plasma and the white membrane layer, as a control example.

[0032] 2. Preparation of solid-phase reaction microplates

[0033] The four cell suspensions prepared in step 1 were separated into 100 μL of each of the 96-well pre-coated U-shaped microplates using a pipette. The plates were then placed in a 4°C freezer for at least 7 hours. After removal, the plates were washed several times with physiological saline, and the red blood cells were lysed with 1:1 PBS. The plates were washed again, and then lysed with freeze-drying protectant to prepare dried solid-phase detection microplates. The solid-phase detection microplates prepared using the red blood cell adhesion enhancers from Examples 1-3 were labeled as Solid-phase Detection Microplate No. 1, Solid-phase Detection Microplate No. 2, and Solid-phase Detection Microplate No. 3, respectively; the solid-phase detection microplate prepared using physiological saline was labeled as Solid-phase Detection Microplate No. 4.

[0034] Experiments were conducted on microplates for solid phase detection, numbers 1-4.

[0035] Experiment 1: Microscopic observation of the distribution and quantity of shadow cells after erythrocyte lysis in microplates 1-4 for solid-phase detection. Specific results are as follows:

[0036] In the No. 1 solid-phase detection microplate, the number of shadow cells formed after erythrocyte lysis is relatively large, and they are evenly and densely distributed in the center and edge of the wells, without any missing corners or eccentric or lateral distribution, which is quite ideal. Figure 1 A)

[0037] In the No. 2 solid-phase detection microplate, the distribution of shadow cells formed after erythrocyte lysis was relatively uniform, present in both the center and edges of the wells, but the density was still relatively low. Figure 1 B);

[0038] The number of shadow cells formed after erythrocyte lysis on microplate No. 3 was acceptable, but the distribution was uneven, with a lateral distribution phenomenon. Figure 1 C);

[0039] In the No. 4 solid-phase detection microplate, the number of shadow cells formed after erythrocyte lysis was relatively small, and their distribution was acceptable. Figure 1 D).

[0040] Experiment 2: Anti-Fya plasma with an agglutination strength of 2+ as determined by the microcolumn gel card method was tested using solid-phase detection microplates (sizes 1-4), repeated three times. The test results are as follows:

[0041] The No. 2 solid-phase detection microplate can detect anti-Fya, which is equivalent to the microcolumn gel card method. Figure 2 B);

[0042] The No. 3 solid-phase detection microplate can detect anti-Fya, slightly stronger than the microcolumn gel card method, but it has a corner-missing phenomenon. Figure 2 C);

[0043] The No. 1 solid-phase detection microplate can detect anti-Fya, and the detection strength is relatively strong, stronger than the microcolumn gel card method. Figure 2 A);

[0044] The No. 4 solid-phase detection microplate can detect anti-Fya, but it is weaker than the microcolumn gel card method. Figure 2 D);

[0045] Based on the results of Experiments 1 and 2, it can be concluded that the effect of suspending red blood cells in microplates 1-3 for solid-phase detection is stronger than that in microplate 4, with microplate 1 showing the best effect. That is, when red blood cells are suspended by the red blood cell adhesion enhancer disclosed in this invention, compared to suspension using physiological saline, it promotes the adhesion of red blood cells to the wells of the microplate.

[0046] The performance of both is comprehensively evaluated below using microplate No. 1 (test plate) and microplate No. 4 (control plate) for solid phase detection.

[0047] 1. Testing of dose-effect antibodies using test and control plates.

[0048] The titers of dose-dependent Kidd blood group system antibodies (anti-JKb) and Duffy blood group system antibodies (anti-Fya) were determined using a microcolumn gel card method. Subsequently, titers were tested using prepared solid-phase agglutination assay plates and control plates. By comparison, the differences in reaction sensitivity between reaction plates prepared with erythrocyte adhesion enhancers and those prepared with erythrocytes suspended in physiological saline were obtained.

[0049] 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, while 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 on the control panel were 64 and 32, respectively. Figure 3 B) The titers were all lower than those measured by the test plate. This indicates that the test plate is superior to the control plate and microcolumn gel card method for detecting dose-effect antibodies.

[0050] 2. Stability testing of experimental and control plates

[0051] Three batches of solid-phase agglutination reaction test plates were prepared by suspending red blood cells with red blood cell attachment enhancer. Simultaneously, three batches of control plates were prepared by suspending red blood cells with physiological saline. Anti-JKb and anti-Fya, which were 2+ on the card, were tested at 1 month, 3 months, 5 months and 7 months of storage, respectively. The focus was on whether there was any change in agglutination intensity and to compare the difference in reaction intensity between the test plates and the control plates at each storage period.

[0052] The experimental results showed that the three batches of solid-phase agglutination reaction test plates prepared from erythrocytes suspended in erythrocytes with erythrocyte adhesion enhancer maintained stable agglutination intensity during the 7-month observation period, with differences not exceeding 1+, and clear distinction between positive and negative controls. Figure 4A); Three batches of control plates prepared from physiological saline-suspended red blood cells showed poor stability in agglutination intensity during the 7-month observation period, with differences reaching 2+, clearly indicating positive and negative controls ( Figure 4 B). Overall, the solid-phase agglutination reaction test plates prepared with erythrocytes suspended in erythrocytes using erythrocyte adhesion enhancers showed stronger agglutination intensity than the control plates prepared with erythrocytes suspended in physiological saline during the same period. After storage for 5 and 7 months, the difference in agglutination intensity between the test plates and the control plates for detecting relevant antibodies was approximately 2+, demonstrating the superior advantage of solid-phase microplates prepared with erythrocyte adhesion enhancers in detecting dose-effect antibodies.

[0053] 3. Precision testing of the test and control plates

[0054] Three batches of solid-phase agglutination reaction test plates prepared by suspending red blood cells with red blood cell attachment enhancer were tested on 94 samples (47 positive and 47 negative) at different times, in different laboratories, and by different operators. The results of the three batches of solid-phase reaction test plates were compared to assess the consistency of the results.

[0055] Test results showed that the test results obtained by the test plates were completely consistent in different laboratories and by different operators, with no false positives or false negatives, indicating that the precision of the test plates was good. In the control plates of the synchronous parallel test, the test results after 7 months of storage were different from those after 1 month and 3 months. The agglutination intensity of positive samples was generally reduced by one agglutination intensity, indicating that the precision of the control plates was not good.

[0056] 4. Test plates and control plates for serum samples

[0057] (1) Ninety-four positive serum samples containing irregular antibodies, whose specificity was determined by the microcolumn gel card method, were tested using test plates and control plates respectively, and the results were compared with those of the microcolumn gel card method.

[0058] (2) Ninety-four normal negative serum samples that were confirmed by the microcolumn gel card method to be free of irregular antibodies were tested using test plates and control plates respectively, and the results were compared with those of the microcolumn gel card method.

[0059] The test results of the test plate and control plate for positive and negative specimens are shown in Table 2. Among the specimens that tested positive in the test plate, 8 specimens tested negative in the control plate. The chi-square statistical results showed that the difference between the two was statistically significant (X). 2 =8, P<0.005), indicating that the serum test results of the experimental plate were better than those of the control plate.

[0060] Table 2. Detection results of serum samples using the test and control plates.

[0061]

Claims

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

2. The red blood cell adhesion enhancer of claim 1, wherein Each 1000 mL of the erythrocyte adhesion enhancer comprises the following components: sodium chloride 1.0 g, disodium ethylenediaminetetraacetate 4.5 g, sodium citrate 1.5 g, and the balance is ultrapure water.

3. The red blood cell adhesion enhancer of claim 1, wherein Each 1000 mL of the erythrocyte adhesion enhancer comprises the following components: sodium chloride 1.0 g, disodium ethylenediaminetetraacetate 3.0 g, sodium citrate 2.0 g, and the balance is ultrapure water.

4. The red blood cell adhesion enhancer of claim 1, wherein Each 1000 mL of the erythrocyte adhesion enhancer comprises the following components: sodium chloride 1.0 g, disodium ethylenediaminetetraacetate 5.0 g, sodium citrate 2.0 g, and the balance is ultrapure water.

5. A method for preparing the red blood cell adhesion enhancer as claimed in any one of claims 1 to 4, characterized by, The preparation procedure is as follows: sodium chloride, disodium ethylenediaminetetraacetate, and sodium citrate are added into ultrapure water and stirred to uniformly dissolve, and then filtered with a 0.22 μm filter to obtain the erythrocyte adhesion enhancer.

6. The erythrocyte adhesion enhancer according to any one of claims 1-4 is used for preparing a solid-phase agglutination reaction microplate for detecting erythrocyte antibodies.

7. Use according to claim 6, wherein In the preparation of the solid-phase agglutination reaction microplate, the erythrocyte adhesion enhancer and human erythrocytes are used to prepare a cell suspension, and then the cell suspension is dispensed into the microplate wells which have been subjected to coating treatment by using a dispensing gun.

8. Use according to claim 7, wherein the compound is ###0002### The cell suspension is prepared by mixing the erythrocyte adhesion enhancer and human packed erythrocytes at a volume ratio of 99.4:0.6.

Images