Acid-eluent reagents for rapid elution of antibodies adsorbed on erythrocyte membranes and their applications

Abstract

This invention relates to an acid eluent reagent for rapidly eluting antibodies adsorbed on erythrocyte membranes and its application. It comprises a 10x concentrated erythrocyte washing buffer, an acid eluent, and a neutralizing solution. The 10x concentrated erythrocyte washing buffer includes the following components: sodium dihydrogen phosphate monohydrate, disodium hydrogen phosphate, bovine serum albumin, xylitol, mannitol, and purified water. The acid eluent includes the following components: glycine and purified water. The neutralizing solution includes the following components: tris(hydroxymethyl)aminomethane, sodium chloride, EDTA·2Na, bovine serum albumin, bromothymol blue, and purified water; the purified water is distilled water or deionized water. This invention can rapidly dissociate antigens adsorbed on cell membranes. The strong acid eluent neutralizing solution is used for antibody recovery for subsequent antibody research; the weak acid eluent effectively maintains the integrity of the cell membrane for subsequent antigen identification.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and medical testing technology, specifically to an acid eluent that rapidly elutes antibodies adsorbed on the erythrocyte membrane and its application. It can rapidly lyse antibody-sensitized erythrocytes and extract free antibodies. This acid eluent is used in laboratory serological methods to study immune-mediated erythrocyte destruction factors. Background Technology

[0002] In the daily work of blood transfusion laboratories, we often encounter "difficult cases" such as weakened antigens or antibodies, unexpected antibody interference, subtypes, and autoimmune hemolytic anemia, which cannot be immediately interpreted and require complex methods to determine the results. A positive direct antiglobulin (DAT) test is the most common type of difficult blood type encountered, where antibodies or complement proteins have already attached to the surface of the red blood cells. Drugs, passive antibody acquisition through transfusion of non-ABO-specific plasma products, hematopoietic stem cell transplantation, monoclonal antibody therapy, Rh immunoglobulins, and allogeneic immune reactions to recent transfusions can all cause a positive DAT test. The positive rate can be as high as 15% in hospitalized patients, seriously affecting patient and transfusion safety. A positive DAT test on neonatal red blood cells indicates hemolytic disease of the fetus and newborn (HDFN). A positive DAT test on adult red blood cells indicates autoimmune hemolytic anemia (AIHA).

[0003] DAT-positive treatment typically employs a three-step acid efflux method. Commonly used methods include cold acid efflux, citric acid efflux, glycine-hydrochloric acid / EDTA efflux, and digitalis saponin-glycine efflux. The basic principle of all these methods is to lower the pH to reduce antigen-antibody binding, thereby dissociating the sensitized IgG antibodies on red blood cells. However, they all share a common drawback: (1) Sample processing before elution: To ensure that the antibodies recovered in the elution solution are from the red blood cell membrane and not unbound "free" antibodies, the red blood cell samples to be eluted must be thoroughly washed before the elution procedure. Wash 4-6 times with physiological saline, pH 7.3 phosphate buffer, or low ionic strength saline (LISS). Sometimes, to help prevent low affinity antibodies from dissociating during washing, the washing solution and reagents need to be pre-cooled at 4°C. This operation is cumbersome and requires real-time monitoring of the temperature. However, the cell membranes of sensitized direct antibody-positive red blood cells or red blood cells that have begun to hemolyze are usually fragile. Repeated washing can cause mechanical damage to the red blood cells, leading to accelerated hemolysis or the shedding of low affinity, low potency sensitized antibodies, resulting in false negative results.

[0004] (2) In the neutralization step, the pH value of the effluent is adjusted to about pH 7.0, or litmus reagent or bromocresol purple is used as an indicator, or pH test paper is used. The neutralization solution is added drop by drop while mixing evenly. However, this operation has too many human factors and relies on subjective judgment. Too high or too low pH value will affect the result.

[0005] (3) Using low-acid elution, 80-90% of the bound IgG antibodies can be completely released. However, in a low-acid environment, the cells are severely damaged. If red blood cells are required for subsequent testing, they need to be washed at least 3 times. However, cell membrane deformation, shrinkage, or spikes may lead to non-specific antibody adsorption and false positives during subsequent antigen identification in low-ionic-strength solutions. If a gentler acid elution method is used, generally with a pH of around 5.0, 80% of IgG-sensitized red blood cells can be dissociated by this method. However, during the dissociation process, a small number of cells need to be removed continuously. The removed cells are washed with physiological saline at least 3 times before performing the DAT test until the DAT test result is negative. This method is for dissociating IgG antibodies bound to red blood cells without destroying the red blood cell membrane antigen. The antigen is then used for antigen detection, but the elution solution cannot be used for antibody detection. Summary of the Invention

[0006] The purpose of this invention is to overcome the above-mentioned shortcomings and provide an acid elution reagent for rapidly eluting antibodies adsorbed on the red blood cell membrane. It can be stored at room temperature for 24 months, does not require a special refrigerated and light-protected environment, and can be used as needed without the need for fresh preparation.

[0007] The objective of this invention is achieved as follows: A rapid acid eluent for eluting antibodies adsorbed on erythrocyte membranes, comprising 10x concentrated erythrocyte washing buffer, acid eluent, and neutralization buffer. The 10x concentrated red blood cell washing solution comprises the following components: sodium dihydrogen phosphate monohydrate, disodium hydrogen phosphate, bovine serum albumin, xylitol, mannitol, and purified water; The acid elution solution comprises the following components: glycine and purified water; The neutralizing solution comprises the following components: tris(hydroxymethyl)aminomethane, sodium chloride, EDTA·2Na, bovine serum albumin, bromothymol blue, and purified water; The pure water is distilled water or deionized water.

[0008] Preferably, in the 10x concentrated red blood cell washing solution, the mass ratio of sodium dihydrogen phosphate monohydrate to disodium hydrogen phosphate is 0.3-0.4:1.8-2.1; the mass ratio of disodium hydrogen phosphate to xylitol is 1:1-1.1; and the mass ratio of bovine serum albumin, xylitol, and mannitol is 4.9-5.1:1.8-2.3:13.9-14.9.

[0009] Preferably, the preparation method of the 10x concentrated red blood cell washing solution is as follows: (1) Weigh out sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate, dissolve and bring to volume with pure water, and adjust the pH to 7.3±0.2 with hydrochloric acid or sodium hydroxide; weigh out bovine serum albumin, xylitol and mannitol and add them to step (1), and let them dissolve at room temperature; (3) after they are completely dissolved, filter them sterile.

[0010] Preferably, the acid elution solution is prepared as follows: glycine is weighed, dissolved in pure water and brought to a final volume, the pH is adjusted to 2.0±0.2 with concentrated hydrochloric acid, and then filtered to remove bacteria.

[0011] Preferably, in the neutralizing solution, the mass ratio of tris(hydroxymethyl)aminomethane, sodium chloride, and EDTA·2Na is 4.7-5.1:2-2.2:1, the mass ratio of bovine serum albumin and bromothymol blue is 1:0.03-0.06, and the mass ratio of EDTA·2Na and bovine serum albumin is 2:1.

[0012] Preferably, the neutralizing solution is prepared as follows: (1) Weigh out tris(hydroxymethyl)aminomethane, sodium chloride, and EDTA·2Na, dissolve them in pure water to a final volume, let them dissolve at room temperature, and adjust the pH to 10.0-10.5 with hydrochloric acid or sodium hydroxide; (2) Weigh out bovine serum albumin and bromothymol blue and add them to the solution in step (1), and filter them sterile.

[0013] An application of an acid eluent for rapidly eluting antibodies adsorbed on erythrocyte membranes, used for washing erythrocytes, releasing antibodies, and dissociating antibodies from erythrocytes.

[0014] Preferably, the washing of the red blood cells includes the following steps: (1) Centrifuge at 1000g for 5 minutes to separate and remove plasma; (2) Prepare 10x concentrated red blood cell washing solution as working washing solution: add 9 volumes of physiological saline to 1 volume of concentrated washing solution and mix; (3) Wash red blood cells: add working washing solution and red blood cell hematocrit at a volume ratio of about 8:1, use a disposable pipette to blow up and down or invert, centrifuge at 1000g for 1 minute and remove supernatant; wash 2-3 times in this way until the supernatant is clear.

[0015] Preferably, the antibody evoked antibody includes the following steps: (1) Add 1 ml of washed packed red blood cells and 1 ml of acid elution buffer to a clean test tube; (2) Use a disposable pipette to blow up and down or invert the tube, and centrifuge at 1000g for 1 min; (3) After centrifugation, immediately transfer the supernatant to another clean test tube; (4) Add 0.2 ml of neutralizing solution to the supernatant. At this time, the color of the neutralizing solution will change from blue to gray-green. (5) Centrifuge the neutralized elution solution at 1000g for 1 min to remove the precipitate. The supernatant elution solution is used for antibody detection.

[0016] Preferably, the dissociation of the red blood cell antibodies includes the following steps: (1) Take a clean test tube, take 0.95 ml of acid elution buffer, add 0.05 ml of neutralization buffer, mix well, and at this time, the pH value of the acid elution buffer will change to 3.8 ± 0.5; (2) Add 1 ml of washed red blood cell hematocrit, and use a disposable pipette to blow up and down or invert; (3) Place at room temperature for 2-3 minutes; (4) Add 0.2 ml of neutralization buffer, and at this time, the color of the neutralization buffer will change from blue to gray-green; (5) Centrifuge at 1000 g for 2 min, take out and discard the supernatant, wash 3 times with working washing buffer, and test the treated red blood cells with anti-IgG. After the result is negative, the cells can be used for antigen detection and absorption experiments.

[0017] The beneficial effects of this invention are: By directly using the acid elution buffer or adjusting its pH value beforehand with a neutralizing solution, antibodies adsorbed on the cell membrane can be rapidly dissociated. Direct use of the strong acid in the acid elution buffer releases the antibodies, followed by neutralization with a neutralizing solution, allowing for antibody recovery for subsequent antibody studies. Alternatively, adjusting the pH of the acid elution buffer to 3.8±0.5 beforehand with a neutralizing solution allows for weak acid release, effectively preserving the cell membrane integrity. The dissociated red blood cells are then used for subsequent antigen identification or absorption experiments. Detailed Implementation

[0018] This invention relates to an acid eluent for rapidly eluting antibodies adsorbed on red blood cell membranes, comprising 10x concentrated red blood cell washing buffer, acid eluent, and neutralizing solution.

[0019] The 10x concentrated red blood cell washing solution comprises the following components: sodium dihydrogen phosphate monohydrate, disodium hydrogen phosphate, bovine serum albumin, xylitol, mannitol, and purified water, wherein the purified water is distilled water or deionized water.

[0020] The mass ratio of sodium dihydrogen phosphate monohydrate to disodium hydrogen phosphate is 0.3-0.4:1.8-2.1; the mass ratio of disodium hydrogen phosphate to xylitol is 1:1-1.1; and the mass ratio of bovine serum albumin, xylitol, and mannitol is 4.9-5.1:1.8-2.3:13.9-14.9. The mass of bovine serum albumin, xylitol, and mannitol in the washing solution is strictly controlled. The addition of bovine serum albumin increases the lubricity of the washing solution, avoiding cell membrane damage during washing, and provides a cationic environment to prevent premature dissociation of low-affinity antigens and antibodies. The addition of mannitol stabilizes the cell membrane, inhibits cell membrane swelling and rupture caused by osmotic pressure imbalance, prevents hemolysis, and reduces the viscosity of the washing solution and scavenge free radicals (antioxidant). The addition of xylitol is particularly effective for fragile and easily hemolyzed red blood cells (such as those from certain AIHA patients), significantly reducing free hemoglobin during washing and increasing the red blood cell recovery rate by about 5%, without interfering with subsequent experiments.

[0021] The preparation method of the 10x concentrated red blood cell washing solution is as follows: (1) Weigh 0.355g sodium dihydrogen phosphate monohydrate and 1.907g disodium hydrogen phosphate, dissolve them in distilled water or deionized water, bring the volume to 100ml, measure the pH, and adjust the pH to 7.3±0.2 with hydrochloric acid or sodium hydroxide; (2) Weigh 5g bovine serum albumin, 2g xylitol and 14.5g mannitol and add them to step (1), and let them dissolve at room temperature; (3) After complete dissolution, use a 0.22μm microporous filter for sterilization filtration.

[0022] The acid elution solution comprises the following components: glycine and pure water, wherein the pure water is distilled water or deionized water.

[0023] The preparation method of the acid elution solution is as follows: Weigh 0.75g of glycine and dissolve it in distilled water or deionized water, make up to 100ml, adjust the pH to 2.0±0.2 with concentrated hydrochloric acid, and filter it with a 0.22μm microporous filter for sterilization.

[0024] The neutralizing solution comprises the following components: tris(hydroxymethyl)aminomethane, sodium chloride, EDTA·2Na, bovine serum albumin, bromothymol blue, and purified water, wherein the purified water is distilled water or deionized water.

[0025] The mass ratio of tris(hydroxymethyl)aminomethane, sodium chloride, and EDTA·2Na is 4.7-5.1:2-2.2:1; the mass ratio of bovine serum albumin and bromothymol blue is 1:0.03-0.06; and the mass ratio of EDTA·2Na and bovine serum albumin is 2:1. EDTA·2Na effectively inhibits the complement cascade reaction by strongly chelating free calcium ions, preventing hemolysis of red blood cells during elution and ensuring the acquisition of intact red blood cells for subsequent detection. Some IgG antibodies (such as anti-Jka, anti-Fya, etc.) bind to red blood cells, and the possible formation of IgM antibody complement complexes depends on divalent cations such as calcium ions. EDTA weakens the antigen-antibody binding force by removing these ions, making it easier for antibodies to elute from the surface of red blood cells under low pH conditions. Tris(hydroxymethyl)aminomethane, as a neutralizing buffer, has strong buffering capacity, minimal interference with subsequent experiments, and effectively protects antibody activity. Tris(hydroxymethyl)aminomethane gently and rapidly terminates the denaturing effect of acid on antibodies, unlike other strong alkalis that generate intense heat and damage cells, effectively protecting cell integrity. Furthermore, it exhibits excellent biocompatibility, stability, reliability, and ease of storage. Bromothymol blue's color change ranges from pH 6.0 (yellow) to 7.6 (blue), covering a weakly alkaline region, with a characteristic green intermediate state around pH 7.0. The color change reaction is reversible; over-treatment can be promptly corrected. It is yellow in acidic conditions and blue in alkaline conditions.

[0026] The neutralizing solution is prepared as follows: Weigh 9.6g of tris(hydroxymethyl)aminomethane, 4.2g of sodium chloride, and 2g of EDTA·2Na and dissolve them in distilled water or deionized water. Let them dissolve at room temperature and bring the volume up to 100ml. Adjust the pH to 10.0-10.5 with hydrochloric acid or sodium hydroxide. (2) Weigh 1g of bovine serum albumin and 0.05g of bromothymol blue and add them to the solution in step (1). Use a 0.22μm microporous filter to sterilize and filter.

[0027] An application of an acid eluent for rapidly eluting antibodies adsorbed on erythrocyte membranes, used for washing erythrocytes, releasing antibodies, and dissociating antibodies from erythrocytes.

[0028] The washing of the red blood cells includes the following steps: (1) Centrifuge at 1000g for 5 minutes to separate and remove plasma, removing as much plasma as possible. (2) Prepare the working wash solution by adding 1 volume of concentrated red blood cell washing solution to 9 volumes of physiological saline. If you plan to use it again, it is recommended to store the remaining washing solution in the refrigerator. (3) Wash the red blood cells: Add the working wash solution and the hematocrit of red blood cells at a volume ratio of approximately 8:1. Use a disposable pipette to blow and aspirate 5 times (or invert 5 times), centrifuge at 1000g for 1 minute, and remove the supernatant. Wash 3 times in this manner.

[0029] The antibody release includes the following steps: (1) Add 1 ml of washed packed red blood cells and 1 ml of acid elution buffer to a clean test tube. (2) Use a disposable pipette to blow and aspirate 5 times (or invert 5 times), and centrifuge at 1000g for 1 min. (3) After centrifugation, immediately transfer the supernatant to another clean test tube. (4) Add 0.2 ml of neutralizing solution to the supernatant. At this time, the color of the neutralizing solution will change from blue to gray-green. (5) Centrifuge the neutralized elution solution at 1000g for 1 min to remove the precipitate. The supernatant elution solution is used for antibody detection.

[0030] The antibody dissociation of the red blood cells includes the following steps: (1) Take a clean test tube, add 0.95 ml of acid elution buffer and 0.05 ml of neutralization buffer, mix well. At this time, the pH value of the acid elution buffer will change to 3.8 ± 0.5. (2) Add 1 ml of washed red blood cell hematocrit and use a disposable pipette to blow up and down 5 times (or invert 5 times). (3) Let stand at room temperature for 2-3 minutes, the time should not exceed 3 minutes. (4) Add 0.2 ml of neutralization buffer. At this time, the color of the neutralization buffer will change from blue to gray-green. (5) Centrifuge at 1000g for 2 minutes, take out and discard the supernatant, wash 3 times with working washing buffer, and test the treated red blood cells with anti-IgG. After the result is negative, the cells can be used for antigen detection and absorption experiments.

[0031] In addition to the above embodiments, the present invention also includes other embodiments. All technical solutions formed by equivalent transformation or equivalent substitution should fall within the protection scope of the claims of the present invention.

Claims

1. An acid eluent for rapidly eluting antibodies adsorbed on erythrocyte membranes, characterized in that: Includes 10x concentrated red blood cell wash, acid elution buffer, and neutralization buffer. The 10x concentrated red blood cell washing solution comprises the following components: sodium dihydrogen phosphate monohydrate, disodium hydrogen phosphate, bovine serum albumin, xylitol, mannitol, and purified water; The acid elution solution comprises the following components: glycine and purified water; The neutralizing solution comprises the following components: tris(hydroxymethyl)aminomethane, sodium chloride, EDTA·2Na, bovine serum albumin, bromothymol blue, and purified water; The pure water is distilled water or deionized water.

2. The acid eluent for rapidly eluting antibodies adsorbed on erythrocyte membranes according to claim 1, characterized in that: In the 10x concentrated red blood cell washing solution, the mass ratio of sodium dihydrogen phosphate monohydrate to disodium hydrogen phosphate is 0.3-0.4:1.8-2.1; the mass ratio of disodium hydrogen phosphate to xylitol is 1:1-1.1; and the mass ratio of bovine serum albumin, xylitol, and mannitol is 4.9-5.1:1.8-2.3:13.9-14.

9.

3. The acid eluent for rapidly eluting antibodies adsorbed on erythrocyte membranes according to claim 1 or 2, characterized in that: The preparation method of the 10x concentrated red blood cell washing solution is as follows: (1) Weigh out sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate, dissolve and bring to volume with pure water, and adjust the pH to 7.3±0.2 with hydrochloric acid or sodium hydroxide; weigh out bovine serum albumin, xylitol and mannitol and add them to step (1), and let them dissolve at room temperature; (3) after they are completely dissolved, filter them sterile.

4. The acid eluent for rapidly eluting antibodies adsorbed on erythrocyte membranes according to claim 1, characterized in that: The preparation method of the acid elution solution is as follows: weigh glycine, dissolve it in pure water and make up the volume, adjust the pH to 2.0±0.2 with concentrated hydrochloric acid, and filter it for sterilization.

5. The acid eluent for rapidly eluting antibodies adsorbed on erythrocyte membranes according to claim 1, characterized in that: In the neutralization solution, the mass ratio of tris(hydroxymethyl)aminomethane, sodium chloride, and EDTA·2Na is 4.7-5.1:2-2.2:1, the mass ratio of bovine serum albumin and bromothymol blue is 1:0.03-0.06, and the mass ratio of EDTA·2Na and bovine serum albumin is 2:

1.

6. The acid eluent for rapidly eluting antibodies adsorbed on erythrocyte membranes according to claim 1 or 5, characterized in that: The neutralizing solution is prepared as follows: (1) Weigh out tris(hydroxymethyl)aminomethane, sodium chloride, and EDTA·2Na, dissolve them in pure water to a final volume, let them dissolve at room temperature, and adjust the pH to 10.0-10.5 with hydrochloric acid or sodium hydroxide; (2) Weigh out bovine serum albumin and bromothymol blue and add them to the solution in step (1), and filter them sterile.

7. The application of an acid eluent for rapidly eluting antibodies adsorbed on erythrocyte membranes, characterized in that: Used for washing red blood cells, releasing antibodies, and dissociating antibodies from red blood cells.

8. The application of the acid eluent for rapidly eluting antibodies adsorbed on erythrocyte membranes according to claim 7, characterized in that: The washing of the red blood cells includes the following steps: (1) Centrifuge at 1000g for 5 minutes to separate and remove plasma; (2) Prepare 10x concentrated red blood cell washing solution as working washing solution: add 9 volumes of physiological saline to 1 volume of concentrated washing solution and mix; (3) Wash red blood cells: add working washing solution and red blood cell hematocrit at a volume ratio of about 8:1, use a disposable pipette to blow up and down or invert, centrifuge at 1000g for 1 minute and remove supernatant; wash 2-3 times in this way until the supernatant is clear.

9. The application of the acid eluent for rapidly eluting antibodies adsorbed on erythrocyte membranes according to claim 7 or 8, characterized in that: The antibody release includes the following steps: (1) Add 1 ml of washed packed red blood cells and 1 ml of acid elution buffer to a clean test tube; (2) Use a disposable pipette to blow up and down or invert the tube, and centrifuge at 1000g for 1 min; (3) After centrifugation, immediately transfer the supernatant to another clean test tube; (4) Add 0.2 ml of neutralizing solution to the supernatant. At this time, the color of the neutralizing solution will change from blue to gray-green. (5) Centrifuge the neutralized elution solution at 1000g for 1 min to remove the precipitate. The supernatant elution solution is used for antibody detection.

10. The application of the acid eluent for rapidly eluting antibodies adsorbed on erythrocyte membranes according to claim 7 or 8, characterized in that: The antibody dissociation of the red blood cells includes the following steps: (1) Take a clean test tube, take 0.95 ml of acid elution buffer, add 0.05 ml of neutralization buffer, mix well, and at this time, the pH value of the acid elution buffer will change to 3.8 ± 0.5; (2) Add 1 ml of washed red blood cell hematocrit, and use a disposable pipette to blow up and down or invert; (3) Place at room temperature for 2-3 minutes; (4) Add 0.2 ml of neutralization buffer, and at this time, the color of the neutralization buffer will change from blue to gray-green; (5) Centrifuge at 1000 g for 2 min, take out and discard the supernatant, wash 3 times with working washing buffer, and test the treated red blood cells with anti-IgG. After the result is negative, the cells can be used for antigen detection and absorption experiments.