Immune magnetic microsphere for separating red blood cells as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of preparation of immunomagnetic beads, and particularly relates to immunomagnetic microspheres for separating red blood cells as well as a preparation method and application of the immunomagnetic microspheres. The immunomagnetic microsphere for separating red blood cells comprises a magnetic microsphere and an anti-red blood cell antibody, and the magnetic microsphere is coated with the anti-red blood cell antibody; the magnetic microspheres are magnetic particle microspheres which are not modified; the magnetic particle microspheres are any one of ferroferric oxide, ferric oxide, ferrous oxide, iron powder or ferrotitanium powder; the mass ratio of the anti-erythrocyte antibody to the magnetic microspheres is 1: (20-400). According to the invention, paramagnetic particle substances without active groups are used, and the paramagnetic particle substances are wide in source and very low in cost, so that compared with the prior art, the production cost of the blood cell separation freeze-dried microspheres is remarkably reduced, the iron content is high, and the magnetic separation time is short.

Description

Technical Field

[0001] The invention belongs to the technical field of immunomagnetic bead preparation, and particularly relates to an immunomagnetic microsphere for separating red blood cells and a preparation method and application thereof. Background Art

[0002] Blood sample testing in clinical examinations requires the patient's blood samples to be separated into serum or plasma according to the requirements of the test items to meet the test needs. The main reason is that the presence of red blood cells will interfere with the antibody capture antigen of the immunological detection method, and the rupture of red blood cells will cause a large amount of intracellular hemoglobin, enzymes, ions and other cell contents to be released, causing strong interference, greatly reducing the specimen quality and the accuracy of the test. The method of separating red blood cells from whole blood to obtain serum or plasma in clinical tests is mainly centrifugation. In addition to the need for centrifugal equipment, the centrifugation operation may also cause the rupture of blood cells, which will reduce the sample quality, and the centrifugation time is about 5 minutes (sodium citrate anticoagulant whole blood sample, 3000rpm). For many acute disease detection projects such as inflammation, myocardial infarction, etc., rapid and accurate blood sample processing and testing is conducive to gaining a treatment time window for patients.

[0003] With the development of clinical testing technology, immunological testing has gradually become a means for clinicians to judge the condition of the disease. Its core is to meet the rapid diagnosis needs of clinical treatment or home monitoring and obtain accurate test results in a timely manner. Immunomagnetic particle technology couples the coated antibody on the surface of magnetic particles to react with antigens and antibodies, binds to the surface of antigens to form immune complexes, and separates the antigen-antibody-magnetic particle immune complexes under the action of strong magnets to achieve cell screening or remove labeled antigens. By using immunomagnetic particle technology to quickly separate red blood cells to obtain plasma, which is applied to immunological testing, it can better serve clinical diagnosis.

[0004] In order to improve the binding force between magnetic particles and antibodies, the existing technology uses magnetic particles modified with chemical bonds, for example:

[0005] In the prior art, Chinese patent CN200710305363.6 discloses a method for separating red blood cells from whole blood using magnetic microspheres coated with lectin or anti-red blood cell antibodies, but the separation time requires 10 minutes.

[0006] Chinese Patent CN200810244503.8 discloses a method for preparing a freeze-dried whole blood or suspended red blood cell separation reagent on the basis of CN200710305363.6. Although the stability of the separation reagent is improved, the separation time still needs 10 minutes. In these two disclosed patents, the magnetic particles used have a particle size of 10-20 μm. In these two disclosed patents, the magnetic particles are magnetite coated with a layer of polymer, and this polymer can combine with phytohemagglutinin by physical adsorption or chemical bond action, but both the reagent stability and the working efficiency are not high.

[0007] Chinese Patent CN202010128026.X discloses a production method using anti-red blood cell antibodies or lectins chemically conjugated with immunomagnetic particles with a diameter of 1-4 μm. The red blood cell adsorption and separation time in 100 μL of whole blood sample is 1 minute. However, the raw materials are too expensive and the production is complex.

[0008] Chinese Patent CN202010490280.4 discloses an immunomagnetic particle for separating red blood cells, its preparation method and application. The preparation method of the immunomagnetic particle includes: Step S1, activating the magnetic particles with an activator, and the weight ratio of the magnetic particles to the activator is 1:0.1-5; the activation time is 5-30 minutes; Step S2, adding anti-red blood cell antibodies to the activated product obtained in Step S1 and performing ultrasonic treatment; Step S3, adding a blocking agent to the product obtained in Step S2 for blocking, and performing strong magnetic separation to obtain an immunomagnetic particle for separating red blood cells; the magnetic particles are polystyrene microspheres coated with magnetite. The particle size of the magnetic particles is 1-5 μm. This patent uses vacuum drying to prepare freeze-dried immunomagnetic particles. The red blood cell adsorption and separation time in 500 uL of whole blood sample is 2 minutes. However, the volume of the whole blood sample that can be separated by this method is limited and cannot be adjusted flexibly, and the separation time of blood cells is too long. Summary of the Invention

[0009] The present invention aims to provide an immunomagnetic microsphere for separating red blood cells, shorten the red blood cell adsorption and separation time in whole blood samples, improve the detection efficiency, and extend the reagent storage time.

[0010] In order to achieve the above object, the technical solution adopted by the present invention is:

[0011] An immunomagnetic microsphere for separating red blood cells, comprising magnetic microspheres and anti-red blood cell antibodies, the magnetic particles being coated with anti-red blood cell antibodies; the magnetic microspheres being unmodified magnetic particle microspheres; the magnetic particle microspheres being any one of magnetite, hematite, ferrous oxide, iron powder or iron titanate powder; the mass ratio of the anti-red blood cell antibody to the magnetic microsphere is 1:20-400.

[0012] In one preferred embodiment, the diameter of the immunomagnetic microspheres does not exceed 1000 nm.

[0013] In one preferred embodiment, the diameter of the immunomagnetic microspheres is 10 - 1000 nm.

[0014] For immunomagnetic microspheres with a diameter greater than 1000 nm, the adsorption efficiency is insufficient and the processing capacity is weak.

[0015] If the particle size of the immunomagnetic microspheres is too small, on the one hand, there are almost no commercially available products with a particle size less than 10 nm, the cost is relatively high, and when the particle size is too small, the magnetic adsorption time will become longer.

[0016] In one preferred embodiment, the mass ratio of the anti - red blood cell antibody to the magnetic microspheres is 1:20 - 100.

[0017] If the mass ratio of the anti - red blood cell antibody to the magnetic microspheres is too large, the number of effective antibodies on the magnetic particles per unit mass becomes smaller, and the processing capacity will decrease.

[0018] If the mass ratio of the anti - red blood cell antibody to the magnetic microspheres is too small, the antibody input amount for production becomes larger, and the cost will increase significantly.

[0019] In order to increase the magnetism in the present invention, the addition amount of magnetic microspheres is much larger than that in the prior art. By adsorbing a small amount of antibody on the magnetic microspheres per unit mass, the separation of red blood cells is realized. However, since the amount of magnetic microspheres added is much larger than that of the modified magnetic microspheres in the prior art, the actual total addition amount of the antibody is equivalent to that in the prior art. However, since the unit cost of the magnetic microspheres is only one ten - thousandth of that of the modified magnetic microspheres, the addition of the magnetic microspheres in the present invention not only significantly reduces the cost. And since the surface of the modified magnetic microspheres is modified by a large number of chemical bonds, the amount of magnetic substances per unit mass is less, so the magnetism is lower. And because of its high cost, increasing the quantity will cause a significant increase in cost. In contrast, the unmodified magnetic microspheres of the present invention have increased magnetism, thereby improving the separation speed.

[0020] Conventionally, it is considered that smaller - sized magnetic particles, due to better dispersion, have a longer sedimentation adsorption time. As pointed out in Chinese Patent No. 202010490280.4, magnetic particles with a particle size greater than 5 μm are not conducive to antibody conjugation, and magnetic particles with a particle size less than 1 μm are not conducive to strong magnetic separation.

[0021] Under the condition of the same mass of magnetic microspheres, the greater the magnet strength, the faster the separation time. Under the condition of the same - strength magnet, the greater the mass of the magnetic particles, the shorter the separation time. In the present invention, under the optimized mass ratio of the antibody and the magnetic microspheres, the small - sized magnetic microspheres (≤1 μm) have a larger specific surface area, have a higher antibody loading capacity, better dispersion, and show more excellent adsorption efficiency for red blood cells and strong magnetic separation efficiency.

[0022] In one preferred embodiment, the immunomagnetic microspheres further comprise a buffer solution containing 1-4 wt% bovine serum albumin. 1-4 wt% bovine serum albumin is the main component in the buffer solution. The inclusion of bovine serum albumin in the immunomagnetic microspheres can promote the dissolution of the freeze-dried microspheres of immunomagnetic particles.

[0023] The immunomagnetic microspheres are prepared by liquid nitrogen freezing and vacuum drying. The present invention uses liquid nitrogen freezing and vacuum drying, which can control the specific quality of the prepared immunomagnetic microspheres, facilitating direct quantitative addition. Each immunomagnetic microsphere has a fixed mass, and the mass of the added immunomagnetic microspheres can be controlled by controlling the specific number or volume of the added immunomagnetic microspheres. The immunomagnetic microspheres prepared by the present invention can be stored at high temperature for 30 days and at room temperature for 2 years.

[0024] The present invention also claims to protect a method for preparing immunomagnetic microspheres for separating red blood cells, which comprises the following steps:

[0025] S1. Add a buffer solution and an anti-red blood cell antibody to the magnetic microspheres and stir evenly. The mass ratio of the anti-red blood cell antibody to the magnetic particles is 1:20 - 400;

[0026] S2. Perform strong magnetic separation on the product obtained in S1, add a frozen buffer solution, mix evenly, and then perform liquid nitrogen freezing and vacuum drying to obtain the immunomagnetic microspheres.

[0027] After strong magnetic separation, a supernatant and a precipitate are obtained. Skim off the supernatant and add a frozen buffer solution to the precipitate.

[0028] Liquid nitrogen freezing is a conventional operation, that is, dropping the liquid droplets into liquid nitrogen (-196 °C) for rapid freezing. On the one hand, it can quickly freeze the liquid droplets, and at the same time, it can also quickly form a shape to form frozen reagent liquid droplets. Vacuum freeze-drying is the operation after liquid nitrogen freezing. Its main purpose is to sublimate the crystal water in the liquid nitrogen frozen reagent liquid droplets, because the liquid reagent balls containing water will quickly melt when entering room temperature and form a liquid again. The differences in the technical parameters of vacuum freeze-drying mainly depend on the technical requirements of different machines, but the general process is: precooling, vacuum freeze-drying, and recovery. Finally, the successful freezing is to test the water content of the formed reagent microspheres, and the water content is less than 5%.

[0029] In one preferred embodiment, the step of liquid nitrogen freezing and vacuum drying is to drop the mixed liquid into liquid nitrogen. The volume of the liquid droplets is 10 - 50 μL to prepare liquid nitrogen frozen microspheres. The vacuum degree is 1 - 10 Pa, the temperature is -50 - -30 °C, and the time is 20 - 40 h.

[0030] In one preferred embodiment, adding a buffer solution and anti-red blood cell antibodies to magnetic microspheres and stirring evenly includes: first adding the buffer solution to the magnetic microspheres to prepare a magnetic microsphere suspension with a concentration of 50-100 mg / mL, and then adding 0.2-5 mg of anti-red blood cell antibodies per milliliter of the suspension and stirring evenly; the buffer solution contains 1-4 wt% bovine serum albumin.

[0031] In one preferred embodiment, the weight ratio of magnetic microspheres to bovine serum albumin is 1 mg: 0.025-0.05 mg.

[0032] In one preferred embodiment, the magnetic microspheres are washed before adding bovine serum albumin first, and the washing includes the following steps: washing the magnetic microspheres with deionized water and then performing strong magnetic separation, discarding the supernatant, and repeating 3 times to obtain magnetic microsphere precipitate.

[0033] In one preferred embodiment, the magnetic microspheres and anti-red blood cell antibodies are suspended at 23-26 °C for 16-20 h for physical adsorption reaction.

[0034] In one preferred embodiment, the buffer solution is NaHCO3, PBS or MES, the concentration of the buffer solution is 0.3-0.7 M, and the pH is 5-9.6. Preferably, the NaHCO3 buffer solution is used for physical adsorption.

[0035] In one preferred embodiment, strong magnetic separation means placing the reaction system in an electromagnetic field or on a magnetic plate for magnetic attraction.

[0036] The present invention also claims to protect another preparation method of immunomagnetic microspheres for separating red blood cells, which includes the following steps:

[0037] S1. Wash the magnetic microspheres 3 times with deionized water, add anti-red blood cell antibodies and a buffer solution to the magnetic microspheres and stir evenly. The mass ratio of anti-red blood cell antibodies to magnetic particles is 1:20-400;

[0038] S2. Perform strong magnetic separation on the product obtained in S1, and add a buffer solution and a cross-linking agent and stir evenly. The mass ratio of magnetic microspheres to the cross-linking agent is 1:0.1-0.25;

[0039] S3. Perform blocking on the product obtained in S2, perform strong magnetic separation, and add a frozen buffer solution, and mix evenly to obtain an immunomagnetic microsphere mixture; after freeze-drying, immunomagnetic microspheres are obtained, and the diameter of the immunomagnetic microspheres is 5.0-1000 nm.

[0040] In one preferred embodiment, the steps of liquid nitrogen freezing and vacuum drying are as follows: the mixed liquid is dropped into liquid nitrogen, the volume of the liquid drop is 10-50 μL to prepare liquid nitrogen frozen microspheres, the vacuum degree is 1-10 Pa, the temperature is -50 to -30 °C, and the time is 20-40 h.

[0041] In one preferred embodiment, adding anti-erythrocyte antibody and buffer into the magnetic microspheres and stirring evenly includes: first adding buffer into the magnetic microspheres to make a magnetic microsphere suspension with a concentration of 50-100 mg / mL, then adding 0.2-5 mg of anti-erythrocyte antibody per milliliter of the suspension, and stirring evenly; the buffer contains 1-4 wt% bovine serum albumin.

[0042] In one preferred embodiment, the weight ratio of the magnetic microspheres to bovine serum albumin is 1 mg: 0.05-0.10 mg.

[0043] In one preferred embodiment, the magnetic microspheres are washed before adding bovine serum albumin first, and the washing includes the following steps: washing the magnetic microspheres with deionized water and then performing strong magnetic separation, discarding the supernatant, repeating 3 times to obtain magnetic microsphere precipitate.

[0044] In one preferred embodiment, the magnetic microspheres and anti-erythrocyte antibody are suspended at 23-26 °C for 16-20 h for adsorption reaction.

[0045] In one preferred embodiment, the buffer is NaHCO3, PBS or MES, the concentration of the buffer is 0.3-0.7 M, the pH is 5-9.6, and preferably NaHCO3 buffer is used for adsorption.

[0046] In one preferred embodiment, strong magnetic separation means placing the reaction system in an electromagnetic field or on a magnetic plate for magnetic adsorption.

[0047] In one preferred embodiment, the concentration of the crosslinking agent is 20-50 mg / mL, and the ratio of magnetic microparticles to the crosslinking agent is 1 mg: 0.1-0.25 mg.

[0048] In one preferred embodiment, the magnetic microparticles and the crosslinking agent are suspended at 23-26 °C for 16-20 h to cause a crosslinking reaction between the antibody on the surface of the magnetic microspheres and bovine serum albumin.

[0049] In one preferred embodiment, the blocking agent is at least one of 10-50 mM lysine and 10-50 mM glycine.

[0050] The present invention also claims the application of another kind of immunomagnetic microspheres for separating red blood cells in separating red blood cells in plasma.

[0051] The present invention also claims a method for separating red blood cells from plasma, comprising: adding immunomagnetic microspheres to a whole blood sample, shaking the sample thoroughly, and then placing the sample on a strong magnetic plate for separation.

[0052] In a preferred embodiment, the mass volume ratio of the immunomagnetic microspheres to the whole blood sample is 10 mg / 500 μL or more.

[0053] In one preferred embodiment, the mass volume ratio of the immunomagnetic microspheres to the whole blood sample is not less than 50 mg / 500 μL.

[0054] The present invention is further explained below:

[0055] The prior art that has been disclosed in the early stage is limited to the conventional immunomagnetic particle labeling method. The magnetic particles must use magnetic beads with modified surface structures, which are modified to obtain magnetic beads containing chemical bonds, and then labeled with antibodies through chemical coupling, so that the antibody and magnetic particles are more firmly bound. In the subsequent cleaning process, the antibody will not be lost and will not affect the measurement results. In general immune reactions, if unmodified magnetic beads are used to coat antibodies by physical adsorption, there are many defects, such as low antibody coating efficiency, resulting in unsatisfactory separation effects.

[0056] The magnetic particles of the present invention are used to process whole blood samples, and the physical adsorption strength is sufficient for red blood cell separation. At the same time, there is no washing process during the processing, which will not cause antibody loss.

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] 1. The present invention obtains a freeze-dried reagent that can quickly separate whole blood red blood cells to obtain upper plasma. The iron content is very high. Under the same magnetic field, the magnetic absorption response is fast. Compared with the modified magnetic particles, the magnetic absorption time is greatly shortened to 5 seconds. The fast separation time of the present invention can better gain treatment time for patients and serve immunological detection projects.

[0059] 2. The present invention uses paramagnetic particulate matter without active groups (ferroferric oxide, ferrous oxide, ferrous oxide, iron powder). Due to its wide source and low cost, which is only one ten-thousandth of the modified magnetic particles, the production cost of blood cell separation freeze-dried microspheres is significantly reduced compared with the prior art. Its iron content is very high, and the magnetic separation time is particularly fast.

[0060] 3. The present invention uses physical adsorption and common chemical cross-linking to coat the anti-erythrocyte antibody, which simplifies the production conditions and steps, avoids the use of expensive coupling chemical reagents, and significantly saves production costs and production time.

[0061] 4. The present invention uses magnetic particles with a particle size of 10 - 1000 nm. Due to their small particle size, large specific surface area, high antibody loading capacity, and good dispersibility in liquid, immunomagnetic particles are prepared by adsorbing or cross-linking anti-red blood cell antibodies, significantly improving the efficiency of red blood cell adsorption and separation compared with the prior art.

[0062] 5. The present invention uses a combination of liquid nitrogen freezing and freeze-drying to produce a quantitative freeze-dried immunomagnetic particle reagent. Through an independently developed freezing buffer, the magnetic particles frozen at low temperature are protected from inactivation of active groups, and immunomagnetic particle freeze-dried microspheres with good uniformity and small batch-to-batch differences can be obtained. Moreover, the reagent can be stored at room temperature for 2 years without the need for low-temperature storage equipment.

[0063] 6. The immunomagnetic particle freeze-dried microspheres produced by the present invention can be increased or decreased according to the volume requirement of whole blood samples, serving clinical testing more simply. Especially, it can process whole blood samples as low as 50 μL. For detection items in immunoassay that require less blood samples and are urgent, plasma can be efficiently obtained for testing, greatly improving the timeliness of the test. Detailed Embodiments

[0064] The present invention will be described in detail below with reference to embodiments. It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other.

[0065] Embodiment 1

[0066] 1.1 Preparation of immunomagnetic

[0067] particle freeze-dried microspheres for separating red blood cells by physical adsorption method

[0068] 1.1.1 Cleaning of magnetic particles

[0069] In this embodiment, the magnetic particles are nano-sized magnetite particles with superparamagnetism, rough surfaces, and a larger specific surface area, capable of loading more anti-red blood cell antibodies.

[0070] The original magnetic particles are solid powders. Weigh 200 mg of magnetite particles (particle size 100 nm), add 2 ml of deionized water, roll and mix evenly for 10 minutes, perform strong magnetic separation, discard the supernatant, and repeat three times to obtain magnetic particle precipitates.

[0071] 1.1.2 Adsorption of antibodies by magnetic particles

[0072] Add 0.2 M sodium bicarbonate buffer (pH 9.6) to the magnetic particle precipitate, and adjust the concentration of the magnetic particles to 100 mg / mL by weighing. Add bovine serum albumin to dissolve it to a concentration of 5 mg / mL. Then add anti-red blood cell antibody, and the mass ratio of the antibody to the magnetic particle mass is 1:100. Suspend at room temperature (≈70 rpm) for 16 hours. After the reaction, use strong magnetic separation, discard the supernatant, and obtain the immunomagnetic particle precipitate.

[0073] 1.1.4 Preparation and preservation of the working concentration of immunomagnetic particles for separating red blood cells

[0074] Add the frozen buffer, 25 mM PBS buffer (2 wt% BSA, pH 7.4), to the immunomagnetic particle precipitate obtained at the end of the adsorption reaction, determine the final concentration of the immunomagnetic particles to be 100 mg / mL, roll and mix (≈70 rpm) for 16 hours, and store at 4°C.

[0075] 1.1.5 Preparation of freeze-dried microspheres of immunomagnetic particles for separating red blood cells by liquid nitrogen freezing and vacuum drying

[0076] Drop the immunomagnetic particle suspension for separating red blood cells at a volume of 40 μL per droplet into liquid nitrogen to prepare liquid nitrogen frozen microspheres. Spread the liquid nitrogen frozen microspheres in a freeze dryer and vacuum dry (10 Pa, -37°C) for 30 hours to obtain the freeze-dried immunomagnetic particle freeze-dried microspheres after freeze-drying. The prepared freeze-dried microspheres of immunomagnetic particles for separating red blood cells can be stored at room temperature for 2 years.

[0077] 1.1.6 Technical advantages

[0078] In this example, the anti-red blood cell antibody was combined with magnetic particles by physical adsorption, with simple operation and low cost. A quantitative freeze-dried separation reagent was prepared, which can be stored at room temperature for 2 years without additional low-temperature storage equipment.

[0079] 1.2 Preparation of freeze-dried microspheres of immunomagnetic particles for separating red blood cells by chemical cross-linking method

[0080] 1.2.1 Magnetic particle cleaning

[0081] In this example, the magnetic particles are nanoscale magnetite particles, which have superparamagnetism, a rough surface, a larger specific surface area, and can load more anti-red blood cell antibodies.

[0082] The original magnetic particles are solid powders. Weigh 200 mg of magnetite particles (particle size 100 nm), add 2 ml of deionized water, roll and mix for 10 minutes, perform strong magnetic separation, discard the supernatant, and repeat three times to obtain the magnetic particle precipitate.

[0083] 1.2.2 Magnetic particle antibody adsorption

[0084] Add 0.2M sodium bicarbonate buffer (pH 9.6) to the magnetic particle precipitate, and adjust the concentration of the magnetic particles to 100 mg / mL by weight. Add bovine serum albumin to dissolve, with a concentration of 5 mg / mL. Then add anti-red blood cell antibody, and the mass ratio of the antibody to the magnetic particles is 1:100. Suspend at room temperature (≈70 rpm) for 16 hours. After the reaction, use strong magnetic separation, discard the supernatant, and obtain the immunomagnetic particle precipitate.

[0085] 1.2.3 Antibody cross-linking and coating of magnetic particles

[0086] Add 0.2M sodium bicarbonate buffer (pH 9.6) to the magnetic particle precipitate, and adjust the concentration of the magnetic particles to 10 mg / mL by weight. Add 2 mL of 1.25% glutaraldehyde (dilute 25% glutaraldehyde to 1.25% with 10 mM, pH = 6.8 PBS), and suspend at room temperature (≈70 rpm) for 18 hours.

[0087] 1.2.4 Magnetic particle blocking

[0088] Perform strong magnetic separation on the suspension product of antibody cross-linked and coated magnetic particles, and discard the supernatant. Wash with 4 mL of 0.2M sodium bicarbonate buffer (pH 9.6), perform strong magnetic separation, and discard the supernatant. Repeat three times to obtain the magnetic particle precipitate.

[0089] Add 2 mL of 0.2M sodium bicarbonate buffer (pH 9.6) to the magnetic particle precipitate, then add 1 mL of 0.2M lysine (0.29 g dissolved in 10 mL of purified water), mix well, and suspend at room temperature (≈70 rpm) for 2 hours for blocking. After the reaction, use strong magnetic separation, discard the supernatant, and obtain the immunomagnetic particle precipitate.

[0090] 1.2.5 Preparation and storage of the working concentration of immunomagnetic particles for separating red blood cells

[0091] Add 25 mM PBS buffer (2 wt% BSA, pH 7.4) to the immunomagnetic particle precipitate obtained at the end of the adsorption reaction, determine the final concentration of the immunomagnetic particles to be 100 mg / mL, roll and mix (≈70 rpm) for 16 hours, and store at 4°C.

[0092] 1.2.6 Preparation of freeze-dried microspheres of immunomagnetic particles for separating red blood cells by liquid nitrogen freezing and vacuum drying

[0093] Drop the preservation suspension of immunomagnetic particles for separating red blood cells onto liquid nitrogen at a volume of 40 μL per drop to prepare liquid nitrogen frozen microspheres. Spread the liquid nitrogen frozen microspheres in a freeze-dryer and perform vacuum drying (10 Pa, -37°C) for 30 hours to obtain freeze-dried immunomagnetic particle freeze-dried microspheres after freeze-drying. The prepared freeze-dried microspheres of immunomagnetic particles for separating red blood cells can be stored at room temperature for 2 years.

[0094] 1.2.7 Technical advantages

[0095] In this embodiment, anti - red blood cell antibodies are encapsulated on magnetic microparticles by chemical cross - linking. The operation is simple, the cost is low, the antibody loading efficiency is high, and the binding is more firm. A quantitative freeze - dried separation reagent is prepared, which can be stored at room temperature for 2 years without additional low - temperature storage equipment.

[0096] Example 2

[0097] Separation time test of the freeze - dried microspheres of immune magnetic microparticles for separating red blood cells

[0098] 2.1. Add one (50 μl) of the freeze - dried microspheres of immune magnetic microparticles for separating red blood cells prepared in Example 1 into 0.5 mL of whole blood sample (the anticoagulant is EDTA - K2), shake well to redissolve, place it on a strong magnetic plate, and the upper plasma can be seen after 3 s.

[0099] 2.2. Take 0.5 mL of whole blood sample (the anticoagulant is EDTA - K2) and let it stand for natural stratification. The upper plasma can be seen after 0.5 hour.

[0100] 2.3. Take 0.5 mL of whole blood sample (the anticoagulant is EDTA - K2) and centrifuge it for stratification (3000 rpm). At the same rotation speed, a better separation effect can be achieved with a centrifugation time of 3 - 5 min, and the upper plasma is separated.

[0101] Table 1 Results of separating plasma from whole blood under different separation conditions

[0102]

[0103] Through the above comparative experiments, adding the freeze - dried microspheres of immune magnetic microparticles for separating red blood cells can quickly separate red blood cells to obtain the upper plasma, without the need to provide additional centrifugation equipment, greatly improving the separation efficiency and shortening the time to obtain plasma.

[0104] Example 3

[0105] Comparison of the red blood cell separation efficiency of immune magnetic microparticle microspheres prepared with different feeding ratios of anti - red blood cell antibodies to magnetic microparticles

[0106] According to the method of Example 1, using ferroferric oxide microparticles as raw materials, prepare freeze - dried microspheres of immune magnetic microparticles for separating red blood cells with the mass ratio of red blood cell antibody to immune magnetic microparticles (particle size 100 nm) being 1:20, 1:50, 1:100, 1:200, 1:400 respectively. Take the freeze - dried microspheres of immune magnetic microparticles with different coupling ratios, add them into 500 μL of whole blood sample (the anticoagulant is EDTA - K2) respectively, redissolve and mix well, place them statically on a strong magnetic plate, and record the adsorption blood stratification time and blood cell residue, as shown in Table 2 below. The end standard for recording the adsorption stratification time is that the volume of the upper plasma is visually stable and unchanged.

[0107] Table 2 Results of separating plasma from microspheres produced with different antibody-to-magnetic particle feeding ratios

[0108]

[0109] In the process of binding antibodies to magnetic particles disclosed in the present invention, the highest feeding ratio of anti-red blood cell antibodies to immunomagnetic particle freeze-dried microspheres can reach 1:20. All microspheres can rapidly separate whole blood, showing a very high strong magnetic separation efficiency. A small amount of blood cell residue phenomenon occurs in immunomagnetic particles with a feeding ratio of 1:400, and immunomagnetic particles with a feeding ratio of 1:20 - 1:100 show good red blood cell separation effects.

[0110] Example 4

[0111] Comparison of preparing freeze-dried magnetic particle reagents by first preparing frozen microspheres with liquid nitrogen and then performing freeze-drying and other freeze-drying methods

[0112] 3.1.1 Prepare red blood cell separation immunomagnetic particle freeze-dried microspheres according to Example 1.

[0113] 3.1.2 Prepare magnetic particle freeze-dried reagents by the method disclosed in Chinese Patent Application No. CN200810244503.8:

[0114] 1) Immerse sheet-shaped insoluble polymers (such as glass fiber or non-woven fabric analogs) in red blood cell separation reagents for half an hour to allow the polymers to fully adsorb the red blood cell separation reagents.

[0115] 2) Place the sheet-shaped insoluble polymers with the fully adsorbed red blood cell separation reagents on the tray of a freeze-dryer and perform freeze-drying (-60 °C, freeze for 4 hours + vacuum dry for 24 hours).

[0116] 3) Cut the freeze-dried polymer red blood cell separation reagents obtained into slices with a size of 1 cm2, and individually package each slice with an aluminum foil bag.

[0117] 3.1.3 Prepare immunomagnetic particle freeze-dried reagents by the method disclosed in Chinese Patent Application No. CN202010490280.4

[0118] Ultrasonically mix the immunomagnetic particle storage solution (40 kHz, 6 min), dispense it into 5 mL vials at 0.2 mL / vial, seal it with a gauze, put it into a vacuum freeze-dryer, and perform vacuum freeze-drying at 10 Pa and -50 °C for 12 h.

[0119] 3.1.4 Compare the production methods of immunomagnetic particle freeze-dried reagents by three different methods:

[0120] Regarding the use of the insoluble polymer in 3.1.2, a thin slice of a certain size is finally made. In this method, magnetic microparticles are physically adsorbed on the polymer during the separation process. When added to whole blood for separation, the dispersibility is poor, so the separation time is long, and the operation involves cutting, making the process more complex.

[0121] Regarding the method in 3.1.3, a freeze-dried reagent is obtained by adding a quantitative volume of immunomagnetic microparticle reagent to a vial and performing freeze-drying. Whole blood needs to be added to the vial to achieve separation. However, when the volume of the whole blood sample increases, due to the fixed dose of the separation reagent, the separation effect of this method is poor.

[0122] In the method of the present invention, a liquid drop with a volume of 10 - 50 μL is dropped into liquid nitrogen for freezing to prepare frozen reagent microspheres, and then freeze-dried to prepare freeze-dried immunomagnetic microparticle freeze-dried microspheres. For different volume requirements of less blood samples needed in clinical tests and high blood volume requirements for multiple tests, the quantitative increase or decrease of the freeze-dried reagent microspheres can be carried out, further reducing the complexity of the operation. In addition, usually for immunomagnetic microparticle preparations, operations such as cryogenic freezing and drying will cause agglomeration of magnetic microparticles, resulting in difficulty in resuspension and dispersion, and affecting the chemical activity of the surface active groups of magnetic microparticles. In the present invention, by adding a buffer solution, it is ensured that after being treated by liquid nitrogen freezing and vacuum drying and then reconstituted, its basic activity can still be maintained.

[0123] Example 5

[0124] Comparison of magnetic microparticle particle size on magnetic separation effect

[0125] According to the method in Example 1, using iron oxide particles as raw materials, freeze-dried immunomagnetic microparticles for separating red blood cells with particle sizes of 10 nm, 20 nm, 60 nm, 100 nm, 500 nm, and 1000 nm are respectively prepared. Take freeze-dried immunomagnetic microparticles with different particle sizes and add them to 500 μL of whole blood samples (the anticoagulant is EDTA-K2) respectively. After fully reconstituting and mixing evenly, place them on a strong magnetic plate and record the time of adsorption of blood stratification and the remaining blood cells. The end standard for recording the adsorption stratification time is that the volume of the upper plasma is visually stable and unchanged.

[0126] Table 3 Results of plasma separation by microspheres produced from microparticles with different particle sizes

[0127]

[0128]

[0129] The results showed that the freeze-dried microspheres of immunomagnetic microparticles with small particle sizes (≤1000 nm) had sufficient surface area and antibody loading, and could all rapidly separate whole blood, showing a very high strong magnetic separation efficiency. A small amount of blood cell residue was observed in the immunomagnetic microparticles with particle sizes of 500 nm and 1000 nm, while the immunomagnetic microparticles with particle sizes of 10 - 100 nm showed good red blood cell separation effect.

[0130] Example 6

[0131] Comparison of the effects of magnetic microparticle materials on red blood cell separation

[0132] According to the method disclosed in Example 1, the freeze-dried microspheres of immunomagnetic microparticles for separating red blood cells with a uniform particle size of 100 nm were respectively prepared from materials of magnetite, ferric oxide, ferrous oxide, iron powder, and titanium iron powder. All the iron materials were purchased from Kete New Material Technology Co., Ltd. in Qinghe County. The freeze-dried microspheres of immunomagnetic microparticles with different materials were respectively added into 500 μl of whole blood samples (with EDTA-K2 as anticoagulant), fully re-dissolved and mixed evenly, and then placed statically on a strong magnetic plate. The time for blood adsorption and stratification and the blood cell residue were recorded. The end standard for recording the time of adsorption and stratification was that the volume of the upper plasma was visually stable and unchanged. The obtained results are shown in Table 4.

[0133] Table 4 Results of plasma separation by microspheres produced from different materials

[0134]

[0135]

[0136] The results showed that under the optimized feeding ratio of the present invention, the freeze-dried microspheres of immunomagnetic microparticles of magnetite, ferric oxide, ferrous oxide, iron powder, and titanium iron powder could all rapidly separate whole blood, showing a very high strong magnetic separation efficiency. The immunomagnetic microparticles of magnetite, ferric oxide, and ferrous oxide showed good red blood cell separation effect. A small amount of blood cell residue was observed in the immunomagnetic microparticles of iron powder and iron titanium powder.

[0137] Example 7

[0138] Comparison of the red blood cell separation efficiency of different doses of freeze-dried microspheres of immunomagnetic microparticles for separating red blood cells

[0139] According to the method of Example 1, prepare separated red blood cell immunomagnetic particle freeze-dried microspheres (particle size 100 nm) with a feeding ratio of red blood cell antibody to immunomagnetic particles of 1:100. Respectively take 10 mg, 25 mg, 50 mg, 100 mg (2 * 50 mg), 200 mg (4 * 50 mg) of different masses of separated red blood cell immunomagnetic particle freeze-dried microspheres, and add them to 500 μL of whole blood samples (anticoagulant is EDTA-K2) respectively. Fully re-dissolve and mix evenly, then place them on a strong magnetic plate, and record the time of adsorbed blood stratification and the remaining blood cells. The standard for recording the end point of adsorption stratification time is that the volume of the upper plasma is visually stable and unchanged.

[0140] Table 5 Results of separating plasma with immunomagnetic particle freeze-dried microspheres of different masses

[0141]

[0142] Treat 500 μL of whole blood samples with separated red blood cell immunomagnetic particle freeze-dried microspheres of different masses. All microspheres can quickly separate whole blood, showing a very high strong magnetic separation efficiency. A large amount of blood cell residue will appear in the 10 mg microspheres, and a small amount of blood cell residue will appear in the 25 mg microspheres. The 50 mg, 100 mg, and 200 mg microspheres show good red blood cell separation effects.

[0143] Example 8

[0144] Use the separated red blood cell immunomagnetic particle freeze-dried microspheres produced by the present invention to conduct tests on different blood sample volumes

[0145] Add 1, 3, 5, 10 parts of separated red blood cell immunomagnetic particle freeze-dried microspheres to 50, 100, 300, 500 μL of whole blood samples (anticoagulant is EDTA-K2) respectively, where each part is 10 mg of immunomagnetic particle freeze-dried microspheres. Fully re-dissolve and mix evenly, then place them on a strong magnetic plate, and record the time of adsorbed blood stratification and the remaining blood cells.

[0146] Table 6 Results of treating whole blood of different volumes with immunomagnetic particle freeze-dried microspheres of different masses

[0147]

[0148]

[0149] By proportionally increasing the separated red blood cell immunomagnetic particle freeze-dried microspheres for different blood volumes, the requirements of efficient separation and no blood cell residue can be effectively achieved, and plasma can be quickly and conveniently obtained by separating red blood cells from whole blood. As the volume of whole blood increases, proportionally increasing the immunomagnetic particle freeze-dried microspheres does not lead to a longer separation time. The separation time of the present invention for 100 - 1000 μL of whole blood is within 5 seconds, with a very high separation efficiency.

[0150] Example 9

[0151] Detection by the procalcitonin chemiluminescence detection kit of the present invention

[0152] Centrifuge the same whole blood sample containing procalcitonin (4°C, 1200 g, 5 min), add separated red blood cell immunomagnetic microparticles, fully dissolve and mix them by freeze-drying, magnetically adsorb them on a strong magnetic plate for 5 s, separate the whole blood red blood cells, and take the upper plasma for testing. Use an automatic chemiluminescence analyzer and a supporting procalcitonin (PCT) determination kit (magnetic microparticle chemiluminescence method) to detect the luminescence value signal of the separated plasma sample. Repeat the test 2 times for samples with different treatment methods, and record the luminescence value.

[0153] Table 7 Chemiluminescence detection results of the procalcitonin item

[0154]

[0155] The whole blood sample containing procalcitonin treated by the present invention is basically consistent with the whole blood sample containing procalcitonin treated by the centrifugation method in the detection results of the luminescence value, indicating that the plasma obtained from the whole blood sample treated by the present invention has a good correlation with the plasma obtained by the centrifugation method and can obtain reliable and accurate detection results.

[0156] Example 10

[0157] Verification of the thermal stability of the separated red blood cell immunomagnetic microparticle freeze-dried microsphere reagent

[0158] Place the prepared separated red blood cell immunomagnetic microparticle freeze-dried microspheres (50 μl) at low temperature (2 - 8°C), room temperature (23 - 26°C), and high temperature (37°C) for 7 days, 15 days, and 30 days respectively. Perform the separation and adsorption test of red blood cells from whole blood samples on the freeze-dried reagent microspheres of the same number of days. Add them to 500 μL of whole blood samples (the anticoagulant is EDTA-K2) respectively, fully dissolve and mix them, and place them on a strong magnetic plate, and record the time of blood adsorption and stratification.

[0159] Table 7 Results of whole blood treated with immunomagnetic microparticle freeze-dried microspheres stored under different conditions

[0160]

[0161] The above detection results show that compared with the immunomagnetic microparticle freeze-dried microspheres stored at low temperature, the freeze-dried reagent microspheres placed at room temperature and high temperature can maintain a good separation effect. Especially for the freeze-dried reagent microspheres stored at high temperature, the storage for 30 days does not affect the separation performance of the reagent.

[0162] Example 11

[0163] Verification of the thermal stability of the reagent of immunomagnetic microparticles for separating red blood cells freeze-dried microspheres for the chemiluminescence detection item of procalcitonin (PCT)

[0164] The plasma separated from the whole blood sample in Example 10 was subjected to the chemiluminescence detection of procalcitonin, and the signal was detected using an automatic chemiluminescence immunoassay analyzer, and the results were recorded.

[0165] Table 8 Detection results of the chemiluminescence detection item of procalcitonin

[0166]

[0167] The detection results show that the immunomagnetic microparticles for separating red blood cells produced by the present invention, after being stored in a high-temperature environment for 30 days, the plasma obtained by whole blood separation and subjected to immunological detection has little influence on the detection results. According to the simulation, the immunomagnetic microparticles freeze-dried microspheres produced by the present invention can be stored at room temperature for up to 2 years.

[0168] The content clarified in the above embodiments should be understood that these embodiments are only used to illustrate the present invention more clearly, rather than to limit the scope of the present invention. After reading the present invention, various equivalent forms of modification of these embodiments by those skilled in the art all fall within the scope defined by the appended claims of the present invention.

Claims

1. An immunomagnetic microsphere for separating red blood cells, characterized in that: The invention comprises magnetic microspheres and anti-erythrocyte antibodies, wherein the magnetic microspheres are coated with anti-erythrocyte antibodies; the magnetic microspheres are unmodified magnetic particle microspheres; the magnetic particle microspheres are any one of ferroferric oxide, ferrous oxide, ferrous oxide, iron powder or ferrotitanium powder; and the mass ratio of the anti-erythrocyte antibodies to the magnetic microspheres is 1:20-400.

2. The immunomagnetic microspheres for separating red blood cells according to claim 1, characterized in that: The diameter of the immunomagnetic microspheres does not exceed 1000 nm; preferably, the diameter of the immunomagnetic microspheres is 10-1000 nm.

3. The immunomagnetic microspheres for separating red blood cells according to claim 1, characterized in that: The mass ratio of the anti-erythrocyte antibody to the magnetic microspheres is 1:20-100.

4. The immunomagnetic microspheres for separating red blood cells according to claim 1, characterized in that: The immunomagnetic microspheres also include 1-4 wt% bovine serum albumin.

5. A method for preparing the immunomagnetic microspheres according to any one of claims 1 to 4, characterized in that: It includes the following steps: S1. Wash the magnetic microspheres with deionized water for 3 times, add buffer and anti-erythrocyte antibody to the magnetic microspheres and stir evenly, the mass ratio of anti-erythrocyte antibody to magnetic microparticles is 1:20-400; S2. The product obtained in S1 is subjected to strong magnetic separation, and a frozen buffer solution is added, mixed evenly, and then freeze-dried in liquid nitrogen vacuum to obtain immunomagnetic microspheres.

6. A method for preparing the immunomagnetic microspheres according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1. Wash the magnetic microspheres with deionized water for 3 times, add anti-erythrocyte antibody and buffer to the magnetic microspheres and stir to mix, the mass ratio of anti-erythrocyte antibody to magnetic microparticles is 1:20-400; S2, subjecting the product obtained in S1 to strong magnetic separation, adding buffer and cross-linking agent and stirring evenly, wherein the mass ratio of magnetic microspheres to cross-linking agent is 1:0.1-0.25; S3, sealing the product obtained in S2, performing strong magnetic separation, adding frozen buffer solution, and mixing evenly to obtain an immunomagnetic microsphere mixture; and freeze-drying to obtain immunomagnetic microspheres.

7. Use of the immunomagnetic microspheres for separating red blood cells according to any one of claims 1 to 4 in separating red blood cells from plasma.

8. A method for separating red blood cells from plasma, characterized in that: include: Add the immunomagnetic microspheres as described in any one of claims 1 to 4 to the whole blood sample, shake well and place on a strong magnetic plate for separation.

9. The method according to claim 8, characterized in that The mass volume ratio of the immunomagnetic microspheres to the whole blood sample is above 10 mg / 500 μL.

10. The method according to claim 8, characterized in that The mass volume ratio of the immunomagnetic microspheres to the whole blood sample is not less than 50 mg / 500 μL.

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