Preparation method of blood type detection reagent and blood type detection reagent card

Abstract

The application discloses a blood type detection reagent and a preparation method of a blood type detection reagent card in the technical field of medical detection, and relates to the technical field of medical detection.The blood type detection reagent comprises microsphere gel and gel buffer solution, the gel buffer solution is based on PBS buffer solution, and aqueous silicone oil is added into the PBS buffer solution.The preparation method of the blood type detection reagent card comprises the following steps: preparing PBS buffer solution, gel buffer solution and a gel matrix; adding the gel matrix into a hole column of a microcolumn gel reagent card, and then placing the microcolumn gel reagent card into a centrifuge for centrifugation; and finally, sealing the hole column of the microcolumn gel reagent card for standby.The blood type detection reagent has high specificity, high sensitivity and strong anti-interference property, the preparation method of the blood type detection card is simple, and the blood type detection card is convenient, fast and accurate in blood type detection operation.

Description

Technical Field

[0001] This invention relates to the field of medical testing technology, and in particular to a blood typing reagent and a method for preparing a blood typing reagent card. Background Technology

[0002] Blood transfusion is a crucial medical procedure for saving patients' lives, and accurate blood typing is the first step in ensuring safe transfusion. Currently, there are commercially available testing reagents and test cards for ABO and Rh blood typing. Microbead gel typing is widely used in clinical settings, laboratories, and blood banks for ABO and Rh blood typing. Microbead gel test cards are used for both forward and reverse typing, and can identify blood types of other blood group systems besides ABO and Rh. To improve the sensitivity of ABO and Rh blood typing, a suitable amount of microbead gel and bovine serum albumin (BSA) is usually added to the gel buffer during the preparation of the microbead gel test reagent. This places agglutinated red blood cells at the top or inside of the gel, while non-agglutinated red blood cells pass through the gaps between the gel particles and settle to the bottom. Because BSA has good lubricating properties, it allows non-agglutinated red blood cells to pass smoothly through the gaps between the gel particles under centrifugal force, effectively preventing dragging during testing. Bovine serum albumin is prone to bacterial growth, and even with the addition of preservatives, it is difficult to completely prevent bacterial proliferation and thus affect shelf life. Since bovine serum albumin is derived from cattle, it cannot be 100% free from infectious diseases, and it is also relatively expensive.

[0003] Therefore, in order to address the shortcomings of the aforementioned blood typing technologies, there is an urgent need to develop a blood typing technology that is highly sensitive and specific, and is easy and fast to operate. Summary of the Invention

[0004] In view of the technical problems existing in the prior art, the present invention provides a blood typing reagent and a method for preparing a blood typing reagent card that are highly sensitive, specific, stable in quality, and capable of performing both forward and reverse typing of ABO and Rh blood types.

[0005] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0006] The blood typing reagent provided by the present invention includes microsphere gel and gel buffer. Preferably, the gel buffer is based on PBS buffer and contains aqueous silicone oil.

[0007] Preferably, the volume ratio of the microsphere gel to the gel buffer is 8:3 to 10:3.

[0008] Preferably, the microsphere gel is a cross-linked dextran microsphere gel, and the particle size range of the microsphere gel is 40 to 120 micrometers.

[0009] Preferably, the PBS buffer contains disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, and potassium chloride, wherein the concentration range of disodium hydrogen phosphate is 9–10.5 mM, the concentration range of potassium dihydrogen phosphate is 1.5–2.5 mM, the concentration range of potassium chloride is 2–3 mM, the concentration range of sodium chloride is 130–140 mM, and the pH range of the PBS buffer is 7.2–7.4.

[0010] Preferably, the water-based silicone oil is a polyether-modified silicone oil with a viscosity of 100–300 mPa·s and a temperature of 20–30°C.

[0011] Preferably, the gel buffer contains 2-8 ml of aqueous silicone oil per 100 ml of the PBS buffer, and the pH value of the gel buffer is 6.8-7.2.

[0012] Preferably, an antibody is added to the gel buffer, and the antibody is at least one of anti-A antibody, anti-B antibody, and anti-D antibody.

[0013] Preferably, the volume ratio of the gel buffer to the antibody is 1:1 to 32:1.

[0014] The present invention provides a method for preparing blood typing test kits using the above-mentioned blood typing reagents, comprising the following steps:

[0015] S1: Prepare a PBS buffer containing disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, and potassium chloride. The concentration range of disodium hydrogen phosphate is 9–10.5 mM, the concentration range of potassium dihydrogen phosphate is 1.5–2.5 mM, the concentration range of potassium chloride is 2–3 mM, and the concentration range of sodium chloride is 130–140 mM. The pH range of the PBS buffer is 7.2–7.4.

[0016] S2: Prepare a gel buffer by adding the aqueous silicone oil to the buffer, adding 2-8 ml of aqueous silicone oil per 100 ml of the PBS buffer, and the pH value of the gel buffer is 6.8-7.2;

[0017] S3: Prepare a gel matrix by adding the gel buffer to the microsphere gel, wherein the volume ratio of the microsphere gel to the gel buffer is 8:3 to 10:3;

[0018] S4: Add gel matrix. Using a microcolumn gel reagent card, add 25-30 μL of the gel matrix into the well column of the microcolumn gel reagent card.

[0019] S5: Centrifugation, place the microcolumn gel reagent card into a centrifuge for centrifugation;

[0020] S6: Seal the wells of the microcolumn gel reagent card for later use.

[0021] Preferably, it includes the following steps:

[0022] S20: Prepare a gel buffer containing antibodies. Add an antibody to the gel buffer prepared in step S2. The antibody is at least one of anti-A, anti-B, and anti-D. The volume ratio of the gel buffer to the added antibody is 1:1 to 32:1. Mix to obtain a gel buffer containing antibodies.

[0023] S30: Prepare a gel matrix containing antibodies by adding the antibody-containing gel buffer to the microsphere gel, wherein the volume ratio of the microsphere gel to the antibody-containing gel buffer is 8:3 to 10:3.

[0024] S40: Add antibody-containing gel matrix. Using a microcolumn gel reagent card, add 25-30 μL of the antibody-containing gel matrix to the wells of the microcolumn gel reagent card.

[0025] The beneficial effects of this invention are:

[0026] The blood typing reagent of this invention has high specificity, high sensitivity, and long shelf life. By using aqueous silicone oil instead of bovine serum albumin, the prepared gel buffer has strong antiseptic and lubricating properties. This not only provides better lubrication for unagglutinated red blood cells passing through the microsphere gel gaps, preventing negative carryover during reverse typing and quality control testing, but also improves agglutination titers in both forward and reverse typing. Furthermore, the blood typing reagent card prepared using the method of this invention can accurately perform ABO blood typing (both forward and reverse) and Rh blood typing tests, with a simple and convenient testing procedure. Attached Figure Description

[0027] Figure 1 This is a flowchart illustrating the preparation process of the blood typing reagent card of the present invention.

[0028] Figure 2 This is a schematic diagram of blood type testing using the blood type testing reagent card prepared according to the present invention. Detailed Implementation

[0029] To enable those skilled in the art to better understand the technical solutions provided by the present invention, the present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited thereto.

[0030] The blood typing reagent of the present invention includes microsphere gel and gel buffer. The gel buffer is based on PBS buffer and contains aqueous silicone oil.

[0031] Microsphere gels act as molecular sieves in blood typing. They can block agglutinations of red blood cells and antibodies at the top or inside of the gel, while allowing unagglutinated red blood cells to pass through the gaps between the microspheres and deposit at the bottom under centrifugal force. If the diameter and average diameter of the microsphere gel are too small, unagglutinated red blood cells will have difficulty passing through the gaps and accumulating at the top or inside of the gel, resulting in false positives. If the diameter and average diameter are too large, the agglutination strength of antibodies and red blood cells will decrease, thus reducing the sensitivity of the blood typing reagent. Cross-linked dextran microsphere gels have a particle size range of 40–120 micrometers and an average particle size of (60 μm). + 5) Micrometers, therefore, cross-linked dextran microspheres with appropriate particle size range are selected for microsphere gels, which can effectively avoid misjudgment and improve aggregation strength.

[0032] When preparing PBS buffer, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, and potassium chloride are mixed. These compounds can be dissolved after mixing as powder, or their solutions can be mixed. The PBS buffer should contain the following concentrations: disodium hydrogen phosphate (9–10.5 mM), potassium dihydrogen phosphate (1.5–2.5 mM), potassium chloride (2–3 mM), and sodium chloride (130–140 mM). The pH of the prepared PBS buffer should be 7.2–7.4.

[0033] To improve the lubricity of microsphere gels, a certain concentration of bovine serum albumin (BSA) is typically added to the PBS buffer. Adding preservatives can prevent bacterial growth in the BSA, thus extending its shelf life. Sodium azide is commonly chosen as a preservative, but its high toxicity necessitates strict control. Adding sodium azide can affect the ionic strength of the gel matrix, negatively impacting the sensitivity and specificity of blood typing reagents. Furthermore, improper addition of preservatives can impair lubrication, causing some unagglutinated red blood cells to fail to pass through the gaps between gel particles, resulting in false positives.

[0034] Aqueous silicone oil is a high-molecular-weight compound that dissolves in PBS buffer, increasing the viscosity of the PBS buffer and thus increasing the contact time between antigens and antibodies on red blood cells. This further enhances the agglutination intensity of antigens and antibodies on red blood cells during both forward and reverse typing assays. Additionally, aqueous silicone oil has a lubricating effect, allowing single, non-agglutinated red blood cells to smoothly reach the bottom of the gel under centrifugal force during reverse typing and quality control assays, thereby avoiding false positives. Therefore, using aqueous silicone oil instead of expensive bovine serum albumin not only improves the lubricity of microsphere gels and the agglutination intensity of antigens and antibodies on red blood cells, but also effectively prevents bacterial growth, resulting in a longer shelf life, effectively avoids infectious disease infections, and reduces raw material costs.

[0035] When preparing the gel buffer, PBS buffer is used as a base, and disodium EDTA and aqueous silicone oil are added to the PBS buffer. Disodium EDTA, as an anticoagulant, can effectively prevent fibrinogen from converting into fibrin, thereby effectively preventing the coagulation of red blood cells in the blood sample. If blood is collected using EDTA tubes for blood typing, only aqueous silicone oil can be added to the PBS buffer. The aqueous silicone oil should be a polyether-modified silicone oil with a viscosity of 100–300 mPa·s and a temperature of 20–30°C. When preparing the gel buffer, 2–8 ml of aqueous silicone oil should be added to every 100 ml of PBS buffer, resulting in a gel buffer containing approximately 3%–5% aqueous silicone oil. The pH of the prepared gel buffer should be 6.8–7.2. Generally, the aqueous silicone oil is added to the PBS buffer at room temperature (around 25°C), mixed thoroughly, and then stored.

[0036] Cross-linked dextran was selected as the microsphere gel, and the above-mentioned gel buffer was added to the microsphere gel to prepare the gel matrix. The volume ratio of microsphere gel to gel buffer was 8:3 to 10:3.

[0037] When using the above-mentioned blood typing reagents for ABO and Rh blood typing, add an appropriate amount of the reagent to the test tube, add red blood cells, and then centrifuge. The microsphere gel can block agglutinated red blood cells and antibody aggregates at the top or inside of the gel, while aqueous silicone oil improves the lubricity of the microsphere gel. Under centrifugal force, aqueous silicone oil makes it easier for unagglutinated red blood cells to pass smoothly through the gaps between the microspheres and deposit at the bottom of the microsphere gel, avoiding false positives. Aqueous silicone oil can also appropriately increase the viscosity of the gel buffer, thereby strengthening the agglutination intensity of antigens and antibodies.

[0038] When performing ABO blood typing, antibodies can be added to the gel buffer to obtain an antibody-containing gel buffer. The added antibody is at least one of anti-A, anti-B, or anti-D antibodies, with a gel buffer to antibody volume ratio of 1:1 to 32:1. Microbead gels and antibody-containing gel buffers are mixed thoroughly at a volume ratio of 8:3 to obtain an antibody-containing gel matrix. An appropriate amount of the antibody-containing gel matrix is ​​added to a test tube. If the added red blood cells have the corresponding antigen, the antigen on the red blood cells will bind to the antibody, forming an agglutination at the top or inside of the microbead gel, resulting in a positive test result. If the added red blood cells do not have the corresponding antigen, under centrifugal force, the red blood cells will pass through the gaps between the microbeads and deposit at the bottom of the microbead gel, resulting in a negative test result.

[0039] The prepared gel buffer and blood typing reagents can be packaged separately in reagent bottles, or the microsphere gel and gel buffer can be prepared and packaged in test tubes or poured into several well columns of microcolumn gel cards, and the openings of the well columns can be sealed with aluminum foil.

[0040] When preparing blood typing test cards using the aforementioned blood typing reagents, the present invention first uses a multi-well microcolumn gel card. Microsphere gel, PBS buffer, and aqueous silicone oil can be mixed together in a specific ratio and then added to each well of the microcolumn gel card. Alternatively, microsphere gel can be loaded into each well of the microcolumn gel card first, then a certain amount of PBS buffer can be injected to ensure thorough mixing, followed by the addition of aqueous silicone oil in a specific ratio, and centrifugation to ensure thorough mixing. Antibodies are then added to the centrifuged wells, followed by a suspension of the test subject's red blood cells for positive blood typing. Alternatively, a suspension of red blood cells with a known blood type can be added to the centrifuged wells, followed by the test subject's plasma, for reverse typing. A suspension of the test subject's red blood cells can also be added to the centrifuged wells for quality control testing. Finally, the microcolumn gel card is centrifuged, and the test results are determined by observing whether agglomerates form on the top of the microsphere gel or inside the gel in the wells of the microcolumn gel card.

[0041] This invention uses the above-mentioned detection reagents to prepare a blood typing test kit for ABO / Rh blood typing. The detection reagents are added to the wells of a microcolumn gel card, as shown below. Figure 1 The specific preparation process flow chart is as follows:

[0042] Example 1:

[0043] The specific preparation steps of this invention for preparing ABO / Rh blood typing reagent cards are as follows:

[0044] S1: Prepare a PBS buffer containing disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, and potassium chloride. The concentration range of disodium hydrogen phosphate is 9–10.5 mM, the concentration range of potassium dihydrogen phosphate is 1.5–2.5 mM, the concentration range of potassium chloride is 2–3 mM, and the concentration range of sodium chloride is 130–140 mM. The pH range of the PBS buffer is 7.2–7.4.

[0045] S2: Prepare gel buffer by adding aqueous silicone oil to the above buffer, adding 2-8 ml of aqueous silicone oil per 100 ml of PBS buffer, and obtaining a gel buffer with a pH of 6.8-7.2.

[0046] S3: Prepare the gel matrix by adding the above-mentioned gel buffer to the microsphere gel, with a volume ratio of microsphere gel to gel buffer of 8:3 to 10:3;

[0047] S4: Add gel matrix. Select a 6-well or 8-well microcolumn gel reagent card and add 25-30 μL of the above gel matrix to each well of the microcolumn gel reagent card.

[0048] S5: Centrifugation. Place the microcolumn gel reagent card into a centrifuge and centrifuge at 300g for 5 minutes.

[0049] S6: Seal the wells of the microcolumn gel reagent card with aluminum foil to prepare the blood typing reagent card for later use.

[0050] S7: Continue using step S6 to prepare the blood typing test kit for ABO / Rh blood typing reverse typing and quality control testing. First, tear off or remove the sealing foil from the wells of the column. Add 20–50 μL of A blood type red blood cell suspension to one well, 20–50 μL of B blood type red blood cell suspension to another well, and 20–50 μL of O blood type red blood cell suspension to yet another well. The concentration of each blood type red blood cell suspension should be 0.8%–1.0%, preferably 1%. After adding the red blood cell suspensions to the three wells, 25 μL of the test subject's plasma can be added to each well for ABO / Rh blood typing reverse typing; alternatively, the test subject's plasma can be omitted for ABO / Rh blood typing quality control testing. After adding the plasma, centrifuge the microcolumn gel card at 90g for 10 minutes and observe the results.

[0051] S8: Seal the perforated column of the blood type test reagent card from step S7 with aluminum foil again, and prepare the card for later use.

[0052] After preparing the blood typing test kit in step S6, the wells can be sealed with aluminum foil and shipped directly from the factory. If it is for urgent use in a hospital or laboratory, after preparing the blood typing test kit in step S6, steps S7 and S8 can be completed to prepare a blood typing test kit for ABO / Rh blood typing reverse typing and quality control testing. The wells can then be sealed with aluminum foil for short-term use.

[0053] Example 2:

[0054] The specific preparation steps of the blood typing reagent card for ABO / Rh blood typing in this invention are as follows:

[0055] S1: Prepare a PBS buffer containing disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, and potassium chloride. The concentration range of disodium hydrogen phosphate is 9–10.5 mM, the concentration range of potassium dihydrogen phosphate is 1.5–2.5 mM, the concentration range of potassium chloride is 2–3 mM, and the concentration range of sodium chloride is 130–140 mM. The pH range of the PBS buffer is 7.2–7.4.

[0056] S2: Prepare gel buffer by adding aqueous silicone oil to the above buffer, adding 2-8 ml of aqueous silicone oil per 100 ml of PBS buffer, and obtaining a gel buffer with a pH of 6.8-7.2.

[0057] S20: Prepare a gel buffer containing antibodies. Add an antibody to the gel buffer prepared in step S2. The antibody is at least one of anti-A, anti-B, and anti-D. The volume ratio of the gel buffer to the added antibody is 1:1 to 32:1. Mix to obtain a gel buffer containing antibodies.

[0058] Gel buffers containing anti-A antibodies, anti-B antibodies, and anti-D antibodies can be prepared according to a volume ratio of gel buffer to antibody of 1:1 to 32:1.

[0059] S30: Prepare a gel matrix containing antibodies by adding the above-mentioned gel buffer containing the above-mentioned antibodies to the above-mentioned microsphere gel, wherein the volume ratio of the microsphere gel to the gel buffer containing antibodies is 8:3 to 10:3.

[0060] S40: Add the antibody-containing gel matrix. Select a 6-well or 8-well microcolumn gel reagent card and add 25-30 μL of the antibody-containing gel matrix to the wells of the microcolumn gel reagent card.

[0061] If using 6-well or 8-well microcolumn gel cards, select three wells of the microcolumn gel card. First, add anti-A antibody, anti-B antibody, and anti-D antibody to each well, respectively. Alternatively, add anti-A antibody to the first well, anti-B antibody to the second well, and anti-D antibody to the third well. Then, add gel buffer to the corresponding wells, mixing them at a gel buffer to antibody volume ratio of 1:1 to 32:1 to obtain antibody-containing gel buffer. Finally, add microspheres to the corresponding wells, with a microsphere to antibody-containing gel buffer volume ratio of 8:3 to 10:3. The total volume of microspheres and antibody-containing gel buffer in each well should be 25–30 μL.

[0062] S5: Centrifugation. Place the microcolumn gel reagent card into a centrifuge and centrifuge at 300g for 5 minutes.

[0063] S6: Seal the wells of the microcolumn gel reagent card with aluminum foil to prepare the blood typing reagent card for later use.

[0064] S7: Continue using step S6 to prepare the ABO / Rh blood typing test kit. First, tear off or remove the sealing foil from the wells of the column without added antibodies. Add 20–50 μL of type A red blood cell suspension to one well, 20–50 μL of type B red blood cell suspension to another well, and then add 20–50 μL of type O red blood cell suspension to yet another well. The concentration of each blood type's red blood cell suspension should be 0.8%–1.0%, preferably 1%. After adding the antibodies, centrifuge the microcolumn gel card at 90g for 10 minutes and observe the results.

[0065] S8: Seal the perforated column of the blood type test reagent card from step S7 with aluminum foil again, and prepare the card for later use.

[0066] Similarly, the blood typing test kit can be prepared in step S6, and the wells can be sealed with aluminum foil before shipping. If it is needed urgently in a hospital or laboratory, after preparing the blood typing test kit in step S6, steps S7 and S8 can be completed to prepare the ABO / Rh blood typing test kit, and the wells can be sealed with aluminum foil for short-term use.

[0067] When performing ABO / Rh blood typing, a positive result can be quickly determined by observing red agglomerates on the upper part or inside the microsphere gel in the well column; conversely, a negative result can be quickly determined by observing red agglomerates at the bottom of the microsphere gel. (See attached image.) Figure 2 As shown, in each well of the microcolumn gel reagent column 1, the blood type test result can be determined based on the position of the red blood cells 3 in the upper or lower part of the microsphere gel 2.

[0068] To further verify the judgment results of the blood typing test kits prepared above for ABO / Rh blood typing, the following two sets of comparative experiments were conducted:

[0069] First group of experiments:

[0070] Experiment (I) of this invention:

[0071] Gel buffer formulation: Add 4 ml of aqueous silicone oil to 100 ml of PBS buffer, and the buffer pH value is 7.0.

[0072] Microcolumn gel cards were prepared using 6-well microcolumn gel cards. The specific preparation method is as follows:

[0073] First well column: Add 30 μL of gel matrix containing antibody A. Mix anti-A antibody and gel buffer at a volume ratio of 1:1 to obtain gel buffer containing antibody A. Then mix microsphere gel with gel buffer containing antibody A at a volume ratio of 8:3 to obtain gel matrix containing antibody A.

[0074] Second well column: Add 30 μL of gel matrix containing B antibody. The anti-B antibody and gel buffer are mixed at a volume ratio of 1:1 to obtain a gel buffer containing B antibody. Then, the microsphere gel is mixed with the gel buffer containing B antibody at a volume ratio of 8:3 to obtain a gel matrix containing B antibody.

[0075] Third well column: Add 30 μL of gel matrix containing D antibody. Mix anti-D antibody and gel buffer at a volume ratio of 1:1 to obtain gel buffer containing D antibody. Then mix microsphere gel with gel buffer containing D antibody at a volume ratio of 8:3 to obtain gel matrix containing D antibody.

[0076] For wells four through six: add 30 μL of gel matrix to each well. Mix the microsphere gel with gel buffer at a volume ratio of 8:3 to obtain the gel matrix.

[0077] Place the microcolumn gel card with the added gel matrix into a centrifuge and centrifuge at 300g for 5 minutes. Set aside for later use.

[0078] Whole blood was collected from three volunteers using EDTA tubes to prepare anticoagulated blood. The EDTA tubes were then centrifuged at 1500g for 5 minutes. The supernatant plasma was extracted for later use; the hematocrit of the lower erythrocytes was extracted, and the erythrocytes were resuspended in PBS buffer to prepare a erythrocyte suspension with a concentration of 1%.

[0079] Step 1: Three microcolumn gel cards were used to test three volunteers with different blood types. 50 microliters of each volunteer's red blood cell suspension were added to the first, second, third, and sixth wells of each microcolumn gel card.

[0080] Step 2: Add 50 μL of standard A-type red blood cell suspension to the fourth well and 50 μL of standard B-type red blood cell suspension to the fifth well. The concentration of red blood cells in both wells is 1%.

[0081] Step 3: Add 25 microliters of the volunteer's plasma to the fourth and fifth wells respectively.

[0082] Step 4: Place each microcolumn gel card into a centrifuge, centrifuge at 90g for 10 minutes, and observe the results.

[0083] The blood typing reagent and blood typing reagent card of the present invention were used for testing, and the experimental results are as follows:

[0084] First hole Second hole Third hole Fourth hole Fifth hole Sixth hole Test results Volunteer 1 4+ - 4+ - 3+ - Type A, positive for D. Volunteer 2 - 4+ 4+ 4+ - - Type B, positive for D. Volunteer 3 - - 4+ 4+ 4+ - Type O D positive

[0085] The test results showed that Volunteer 1 was positive for type A RH, and wells 4 and 6 were clean with no residue; Volunteer 2 was positive for type B RH, and wells 5 and 6 were clean with no residue; Volunteer 3 was positive for type O RH, and well 6 was clean with no residue.

[0086] A comparative experiment was conducted to compare the performance of the buffer solutions. A gel buffer was prepared by adding 1.25 g of bovine serum albumin (BSA) and 2.79 g of disodium EDTA to 100 ml of PBS buffer, and adjusting the pH to 7.0 using 5 mol / L sodium hydroxide. Other procedures were the same as described above. The experimental results are as follows:

[0087] First hole Second hole Third hole Fourth hole Fifth hole Sixth hole Test results Volunteer 1 4+ - 4+ - 2+ - Type A, positive for D. Volunteer 2 - 4+ 4+ 4+ - - Type B, positive for D. Volunteer 3 - - 4+ 4+ 4+ - Type O D positive

[0088] Through comparative experiments, this invention demonstrates... Figure 2 As can be seen, in each well 1 of the microcolumn gel reagent, the blood type test result can be determined based on the position of the red blood cells 3 in the upper or lower part of the microsphere gel 2. Additionally, from the attached... Figure 2 It is known that the agglutination strength of the fifth well of blood type A in this invention is slightly stronger than that of the agglutination strength using BSA and EDTA disodium as buffer.

[0089] Second group of experiments:

[0090] Experiment (II) of this invention:

[0091] The experiment to increase agglutination strength for positive blood typing using the blood typing reagent of the present invention is as follows:

[0092] Gel buffer formulation: Add 3 ml of aqueous silicone oil to 100 ml of PBS buffer, and the buffer pH is 7.0.

[0093] Microcolumn gel cards were prepared using 8-well microcolumn gel cards. The specific preparation method is as follows:

[0094] For wells 1 to 4: Add 30 μL of gel matrix containing antibody A. Mix anti-A antibody with gel buffer at a volume ratio of 1:32 to obtain gel buffer containing antibody A. Then mix microsphere gel with gel buffer containing antibody A at a volume ratio of 8:3 to obtain gel matrix containing antibody A.

[0095] Columns in wells 5 through 8: To verify the role of aqueous silicone oil, the gel buffer contained only PBS buffer. 30 μL of gel matrix containing antibody A was added. Anti-A antibody and PBS buffer were mixed at a volume ratio of 1:32 to obtain gel buffer containing antibody A. Then, the microsphere gel was mixed with the gel buffer containing antibody A at a volume ratio of 8:3 to obtain gel matrix containing antibody A.

[0096] Place the microcolumn gel card with the added gel matrix into a centrifuge and centrifuge at 300g for 5 minutes. Set aside for later use.

[0097] Anticoagulated blood was prepared by drawing whole blood from type A volunteers using EDTA tubes. The EDTA tubes were then centrifuged at 1500g for 5 minutes. The hematocrit of the lower layer of red blood cells was extracted, and the red blood cells were resuspended in PBS buffer to prepare a red blood cell suspension with a concentration of 1%.

[0098] Add 50 μL of red blood cell suspension to each well of the column, then place the microcolumn gel card into a centrifuge and centrifuge at 90g for 10 min. Observe the results as follows:

[0099] First hole Second hole Third hole Fourth hole Fifth hole Sixth hole Seventh hole Eighth hole 4+ 4+ 4+ 4+ 3+ 3+ 3+ 3+

[0100] This invention uses aqueous silicone oil to replace expensive bovine serum albumin, thus reducing the cost of blood typing reagents. Because aqueous silicone oil has a certain viscosity, when dissolved in PBS, it increases the viscosity of the PBS, thereby increasing the contact time between red blood cells and antibodies, and thus increasing the agglutination intensity of red blood cells and antibodies. Aqueous silicone oil not only improves the lubricity of the microsphere gel but also effectively prevents bacterial growth, resulting in a longer shelf life and avoiding infection from infectious diseases. The blood typing reagent of this invention has high specificity, high sensitivity, and strong anti-interference properties. Using this blood typing reagent card for blood typing is convenient, fast, and accurate. It does not require high operator skills and can be performed in any location outside of hospitals. The testing procedure is simple and inexpensive.

[0101] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A blood typing reagent, comprising microsphere gels and gel buffer, characterized in that: The gel buffer is based on PBS buffer, which contains aqueous silicone oil. The volume ratio of the microsphere gel to the gel buffer is 8:3 to 10:3; The microsphere gel is a cross-linked dextran microsphere gel, and the particle size range of the microsphere gel is 40-120 micrometers; The PBS buffer contains disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, and potassium chloride. The concentration range of disodium hydrogen phosphate is 9–10.5 mM, the concentration range of potassium dihydrogen phosphate is 1.5–2.5 mM, the concentration range of potassium chloride is 2–3 mM, and the concentration range of sodium chloride is 130–140 mM. The pH range of the PBS buffer is 7.2–7.

4. The water-based silicone oil is a polyether-modified silicone oil with a viscosity of 100–300 mPa·s and a temperature of 20–30°C.

2. The blood typing reagent according to claim 1, characterized in that: The gel buffer is composed of 2-8 ml of the aqueous silicone oil added to every 100 ml of the PBS buffer, and the pH value of the gel buffer is 6.8-7.

2.

3. The blood typing reagent according to claim 2, characterized in that: An antibody is added to the gel buffer, and the antibody is at least one of anti-A antibody, anti-B antibody, and anti-D antibody.

4. The blood typing reagent according to claim 3, characterized in that: The volume ratio of the gel buffer to the antibody is 1:1 to 32:

1.

5. A method for preparing a blood typing reagent card using the blood typing reagent according to any one of claims 1-4, characterized in that, It includes the following steps: S1: Prepare a PBS buffer containing disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, and potassium chloride. The concentration range of disodium hydrogen phosphate is 9–10.5 mM, the concentration range of potassium dihydrogen phosphate is 1.5–2.5 mM, the concentration range of potassium chloride is 2–3 mM, and the concentration range of sodium chloride is 130–140 mM. The pH range of the PBS buffer is 7.2–7.

4. S2: Prepare a gel buffer by adding the aqueous silicone oil to the buffer, adding 2-8 ml of aqueous silicone oil per 100 ml of the PBS buffer, wherein the pH of the gel buffer is 6.8-7.2; S3: Prepare a gel matrix by adding the gel buffer to the microsphere gel, wherein the volume ratio of the microsphere gel to the gel buffer is 8:3 to 10:3; S4: Add gel matrix. Using a microcolumn gel reagent card, add 25-30 μL of the gel matrix into the well column of the microcolumn gel reagent card. S5: Centrifugation, place the microcolumn gel reagent card into a centrifuge for centrifugation; S6: Seal the wells of the microcolumn gel reagent card for later use.

6. The method for preparing the blood typing test kit according to claim 5, characterized in that, It includes the following steps: S20: Prepare a gel buffer containing antibodies. Add an antibody to the gel buffer prepared in step S2. The antibody is at least one of anti-A, anti-B, and anti-D. The volume ratio of the gel buffer to the added antibody is 1:1 to 32:

1. Mix to obtain a gel buffer containing antibodies. S30: Prepare a gel matrix containing antibodies by adding the antibody-containing gel buffer to the microsphere gel, wherein the volume ratio of the microsphere gel to the antibody-containing gel buffer is 8:3 to 10:

3. S40: Add antibody-containing gel matrix. Using a microcolumn gel reagent card, add 25-30 μL of the antibody-containing gel matrix to the wells of the microcolumn gel reagent card.

Images