Lyophilized reagent spheres for immunoassays and methods of making same

Abstract

The application discloses a freeze-dried reagent ball for immunodetection and a preparation method thereof. The preparation method comprises the following steps: mixing specific antibodies, a reaction enhancer, freeze-drying excipients and a surfactant into a mixed solution. The mixed solution is dispersed to form a plurality of liquid droplets with equal volumes, and each liquid droplet is frozen to form a frozen body. The frozen body is freeze-dried to form the freeze-dried reagent ball. The freeze-drying excipients at least comprise trehalose and hydroxypropyl-beta-cyclodextrin. The freeze-dried reagent ball for immunodetection and the preparation method thereof can at least solve the problem of low activity of antibodies in the freeze-dried reagent ball in the prior art.

Description

Technical Field

[0001] This application relates to the field of detection technology, and more specifically, to a lyophilized reagent ball for immunoassay and its preparation method. Background Technology

[0002] In related technologies, when performing immunoassay on samples, microfluidic detection chips can be used to quantify and mix samples and reagents, and the corresponding lyophilized reagent balls for immunoassay can be placed in the microfluidic detection chip so that after the lyophilized reagent balls are reconstituted, they can be mixed again with the sample and reagent mixture to form the solution to be tested.

[0003] However, the lyophilized reagent balls for immunoassay prepared in related technologies have the problem of low antibody activity within the lyophilized reagent balls. Summary of the Invention

[0004] The main objective of this application is to provide a lyophilized reagent ball for immunoassay and its preparation method, in order to solve the problem of low antibody activity in lyophilized reagent balls in related technologies.

[0005] According to one aspect of this application, a method for preparing lyophilized reagent balls for immunoassay is provided, comprising: The specific antibody, reaction enhancer, lyophilization excipient, and surfactant are mixed into a mixed solution; The mixed solution is dispersed into multiple droplets of equal volume, and each droplet is frozen to form a frozen body; The frozen body was freeze-dried to form freeze-dried reagent spheres; The freeze-drying excipients include at least trehalose and hydroxypropyl-β-cyclodextrin.

[0006] In some embodiments, the trehalose concentration in the lyophilized excipient is 2%-5% by mass volume; and / or, The hydroxypropyl-β-cyclodextrin concentration in the lyophilized excipient is 0.5%-2% by mass volume.

[0007] In some embodiments, the step of dispersing the mixed solution into a plurality of equal-volume droplets and freezing each droplet to form a frozen body includes: The mixed solution is used to form multiple droplets of equal volume using a quantitative titration device; The liquid droplets are added to liquid nitrogen to prepare the frozen body.

[0008] In some embodiments, the step of freeze-drying the frozen body to form lyophilized reagent spheres includes: Under a predetermined vacuum level P, the frozen body is frozen for a first predetermined time A1 within a first predetermined temperature T1. The temperature is increased to a second predetermined temperature T2 at a predetermined heating rate V, so that the ice crystals in the frozen body sublimate. The frozen body is heated to a third predetermined temperature T3 at the predetermined heating rate V, and then dried for a second predetermined time A2 to form the lyophilized reagent ball.

[0009] In some embodiments, the predetermined vacuum level P satisfies the relationship: P < 10 Pa; and / or, The first predetermined temperature T1 satisfies the following relationship: -45℃≤T1≤-35℃; and / or, The first predetermined time A1 satisfies the following relationship: 2 hours ≤ A1 ≤ 4 hours; and / or, The predetermined heating rate satisfies the relationship 5℃ / hour ≤ V ≤ 10℃ / hour; and / or, The second predetermined temperature T2 satisfies the following relationship: -15℃ ≤ T2 ≤ -5℃; and / or, The third predetermined temperature T3 satisfies the following relationship: 25℃≤T3≤30℃; and / or, The second predetermined time A2 satisfies the relationship 4 hours ≤ A2 ≤ 8 hours.

[0010] In some embodiments, the reaction enhancer includes at least one of polyethylene glycol 4000 and polyethylene glycol 6000; and / or, The surfactant includes at least one of polysorbate 20 and polysorbate 80; and / or, The specific antibody includes at least one of canine C-reactive protein specific antibody and feline serum amyloid A antibody.

[0011] In some embodiments, the concentration of the specific antibody in the mixed solution is 0.2 mg / mL to 1.5 mg / mL; and / or, In the mixed solution, the mass-volume percentage concentration of the reaction enhancer is 3%-6%; and / or, In the mixed solution, the concentration of the surfactant by mass volume percentage is 0.001%-0.005%.

[0012] In some embodiments, the step following freeze-drying the frozen body to form lyophilized reagent spheres further includes: In a dry, inert gas environment, the lyophilized reagent spheres with a diameter conforming to a predetermined diameter R are selected.

[0013] In some embodiments, the predetermined diameter R satisfies the relationship: 0.5mm ≤ R ≤ 0.8mm; and / or, The gas in the dry inert gas environment includes at least one of nitrogen, helium, neon, argon, krypton, or xenon.

[0014] On the other hand, this application also provides a lyophilized reagent ball for immunoassay, which is prepared by the method described above.

[0015] In this application, during the preparation of lyophilized reagent spheres, the lyophilization excipients in the mixed solution contain at least two components: trehalose and hydroxypropyl-β-cyclodextrin. During the lyophilization process, trehalose forms a glassy matrix around the specific antibody, protecting it and preventing ice crystals generated on the lyophilized reagent spheres from directly contacting and damaging the antibody. Meanwhile, the hydroxypropyl-β-cyclodextrin molecule forms a hydrophobic cavity that encapsulates the hydrophobic regions of the antibody, preventing unfolding of these regions during the lyophilization stage and thus antibody inactivation. Furthermore, after the hydroxypropyl-β-cyclodextrin molecule encapsulates the hydrophobic regions of the antibody, multiple antibodies approach each other during lyophilization, preventing binding between their hydrophobic regions and thus antibody inactivation. In short, the combination of trehalose and hydroxypropyl-β-cyclodextrin results in a glassy matrix coating around the antibody, improving the structural strength of the lyophilized reagent spheres, preventing external substances from affecting the antibody, and enhancing antibody activity. Meanwhile, hydroxypropyl-β-cyclodextrin encapsulates the hydrophobic regions of the antibody, preventing the hydrophobic regions from unfolding or binding between multiple hydrophobic regions, thereby improving the antibody activity to some extent. Attached Figure Description

[0016] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, are illustrative and descriptive, serving to explain this application and do not constitute an undue limitation thereof. In the drawings: Figure 1 This is a schematic diagram of the preparation method of lyophilized reagent balls for immunoassay disclosed in the embodiments of this application. Detailed Implementation

[0017] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0018] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0019] Unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps described in these embodiments do not limit the scope of this application. It should also be understood that, for ease of description, the dimensions of the various parts shown in the drawings are not drawn to actual scale. Techniques, methods, and devices known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, methods, and devices should be considered part of the specification. In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values. It should be noted that similar reference numerals and letters in the following drawings denote similar items; therefore, once an item is defined in one drawing, it need not be further discussed in subsequent drawings.

[0020] As described in the background section, in related technologies, lyophilized reagent beads require freeze-drying during the forming stage. This freeze-drying process typically involves prolonged low-temperature freeze-drying, which can cause the hydrophobic regions of the antibody to unfold or multiple hydrophobic regions to recombine, ultimately leading to antibody inactivation. Furthermore, ice crystals may also form during the freeze-drying stage, directly damaging the antibody and causing inactivation.

[0021] To address the problems existing in the relevant technologies, see [link to relevant documentation]. Figure 1 As shown in the embodiments of this application, a method for preparing lyophilized reagent spheres for immunoassay is provided. The method includes: mixing a specific antibody, a reaction enhancer, a lyophilization excipient, and a surfactant into a mixed solution; dispersing the mixed solution into multiple droplets of equal volume; freezing each droplet to form a frozen body; and freeze-drying the frozen body to form lyophilized reagent spheres. The lyophilization excipient includes at least trehalose and hydroxypropyl-β-cyclodextrin.

[0022] In this application, during the preparation of lyophilized reagent spheres, the lyophilization excipients in the mixed solution contain at least two components: trehalose and hydroxypropyl-β-cyclodextrin. During the lyophilization process, trehalose forms a glassy matrix around the specific antibody, protecting it and preventing ice crystals generated on the lyophilized reagent spheres from directly contacting and damaging the antibody. Meanwhile, the hydroxypropyl-β-cyclodextrin molecule forms a hydrophobic cavity that encapsulates the hydrophobic regions of the antibody, preventing unfolding of these regions during the lyophilization stage and thus antibody inactivation. Furthermore, after the hydroxypropyl-β-cyclodextrin molecule encapsulates the hydrophobic regions of the antibody, multiple antibodies approach each other during lyophilization, preventing binding between their hydrophobic regions and thus antibody inactivation. In short, the combination of trehalose and hydroxypropyl-β-cyclodextrin results in a glassy matrix coating around the antibody, improving the structural strength of the lyophilized reagent spheres, preventing external substances from affecting the antibody, and enhancing antibody activity. Meanwhile, hydroxypropyl-β-cyclodextrin encapsulates the hydrophobic regions of the antibody, preventing the hydrophobic regions from unfolding or binding between multiple hydrophobic regions, thereby improving the antibody activity to some extent.

[0023] In some embodiments, the trehalose concentration in the lyophilized excipient is 2%-5% by mass volume.

[0024] Specifically, when the trehalose concentration (by weight / volume percentage) is below 2%, the resulting lyophilized reagent spheres have a loose framework, poor structural strength, and are prone to collapse. Simultaneously, the formed glassy matrix makes it difficult to prevent ice crystals on the lyophilized reagent spheres from directly contacting the antibodies, leading to easy damage to the antibodies within the lyophilized reagent spheres by the ice crystals. Conversely, when the trehalose concentration (by weight / volume percentage) is greater than 5%, the viscosity of the mixed solution is too high, and during the process of dispersing the mixed solution to form droplets, stringing easily occurs on the droplets, resulting in significant differences in the volume of each droplet. Furthermore, excessively high trehalose content results in fewer pores in the formed lyophilized reagent spheres, leading to excessively long reconstitution times after the lyophilized reagent spheres come into contact with the sample. When the trehalose concentration (by weight / volume percentage) is within the range of 2%-5%, the resulting lyophilized reagent spheres have a regular morphology, good reconstitution performance, and high antibody activity within the lyophilized reagent spheres. In this embodiment, the trehalose concentration (by weight / volume percentage) can be 2%, 3%, 4%, or 5%.

[0025] In some embodiments, the mass-volume percentage concentration of hydroxypropyl-β-cyclodextrin in the lyophilized excipient is 0.5%-2%.

[0026] Specifically, when the mass-volume percentage concentration of hydroxypropyl-β-cyclodextrin is less than 0.5%, the amount of hydroxypropyl-β-cyclodextrin is insufficient, making it difficult to adequately encapsulate the hydrophobic regions of the antibody. Consequently, during the formation of lyophilized reagent spheres, some hydrophobic regions of the antibody still unfold or bind to the hydrophobic regions of other antibodies. Furthermore, lower concentrations of hydroxypropyl-β-cyclodextrin cannot effectively shield against interference from impurities such as proteins and lipid matrices, and the skeletal support structure formed with trehalose is weak, leading to slight shrinkage and irregular surfaces in the lyophilized reagent spheres, resulting in a lower yield. Conversely, when the mass-volume percentage concentration of hydroxypropyl-β-cyclodextrin is greater than 2%, the viscosity of the mixed solution increases significantly, making it difficult to form uniformly sized droplets during dispersion, resulting in poor consistency of the final lyophilized reagent spheres. Additionally, excessive cyclodextrin content can over-encapsulate the antibody, ultimately hindering antibody-antigen binding and causing significant detection errors. Moreover, high concentrations of cyclodextrin themselves produce weak light scattering in turbidimetric systems, increasing blank absorbance and reducing detection sensitivity. On the other hand, although cyclodextrin molecules are hydrophobic internally, they are hydrophilic externally. Excessive cyclodextrin content can cause lyophilized reagent spheres to easily absorb moisture from the air, making them susceptible to moisture absorption during storage. However, when the mass-volume percentage concentration of cyclodextrin is between 0.5% and 2%, the antibody survival rate within the lyophilized reagent spheres is high, and there is no significant error in immunoassay. Furthermore, the resulting lyophilized reagent spheres are uniform in size and volume. In this embodiment, the mass-volume percentage concentration of hydroxypropyl-β-cyclodextrin can be 0.5%, 1%, 1.5%, or 2%.

[0027] In some embodiments, the step of dispersing the mixed solution into a plurality of equal-volume droplets and freezing each droplet to form a frozen body includes: passing the mixed solution through a quantitative titration device to form a plurality of equal-volume droplets; and then dropping the droplets into liquid nitrogen to prepare the frozen body.

[0028] Specifically, the droplets freeze instantly during the infusion of liquid nitrogen, forming small ice crystals that are less likely to puncture the antibody, thus maximizing antibody activity. Simultaneously, trehalose generates a glassy matrix and forms a spherical framework with cyclodextrin to protect the antibody. In this embodiment, the quantitative titration device can be an automatic dropper, meaning that after the mixed solution passes through the automatic dropper, it produces liquid with uniform volume and diameter, ensuring that the prepared lyophilized reagent balls are of uniform volume and size.

[0029] In some embodiments, the step of freeze-drying a frozen body to form lyophilized reagent spheres includes: freezing the frozen body at a first predetermined temperature T1 for a first predetermined time A1 under a predetermined vacuum P; heating the frozen body to a second predetermined temperature T2 at a predetermined heating rate V to sublimate the ice crystals within the frozen body; and heating the frozen body to a third predetermined temperature T3 at a predetermined heating rate V and drying the frozen body for a second predetermined time A2 to form lyophilized reagent spheres.

[0030] Specifically, the first predetermined temperature T1 is lower than the second predetermined temperature T2, and the second predetermined temperature T2 is lower than the third predetermined temperature T3. This keeps the frozen body frozen at the first predetermined temperature T1 for a first predetermined time A1. This process is used to stabilize the skeletal structure formed by trehalose and cyclodextrin in the frozen body, avoiding problems such as collapse, deformation, and cracking caused by rapid heating. The process of heating to the second predetermined temperature T2 at a predetermined heating rate V, causing the ice crystals inside the frozen body to sublimate, uses a higher temperature to ensure the sublimation of the ice crystals inside the frozen body, while preventing the surface of the frozen body from drying out and blocking the water vapor generated inside the frozen body, ensuring thorough sublimation and uniform drying of the internal moisture. The process of heating to the third predetermined temperature T3 at a predetermined heating rate V is to remove bound water from the material at a high temperature, controlling the residual moisture in the lyophilized reagent balls to an extremely low level, significantly improving long-term storage stability and preventing antibody activity attenuation during room temperature storage.

[0031] In some embodiments, the predetermined vacuum level P satisfies the relationship: P < 10 Pa. Specifically, the high vacuum environment ensures that ice crystals can sublimate directly at low temperatures, while reducing the oxygen partial pressure, minimizing oxidative denaturation of antibodies during freeze-drying, and improving the activity retention rate.

[0032] In some embodiments, the first predetermined temperature T1 satisfies the relationship: -45℃ ≤ T1 ≤ -35℃. Specifically, the first predetermined temperature is lower than the collapse temperature of the frozen body, which can stabilize the skeletal structure formed by trehalose and cyclodextrin in the frozen body, prevent the frozen body from collapsing during the heating process, and stabilize the ice crystal structure, laying the foundation for subsequent sublimation. In this embodiment, T1 can be -45℃, -44℃, -43℃, -42℃, -41℃, -40℃, -39℃, -38℃, -37℃, -36℃, or -35℃.

[0033] In some embodiments, the first predetermined time A1 satisfies the relationship: 2 hours ≤ A1 ≤ 4 hours. Specifically, in a prolonged low-temperature environment, the temperature throughout the frozen body remains almost uniform, and the prolonged low temperature improves the stability of the skeletal structure. In this embodiment, the first predetermined time can be 2 hours, 3 hours, or 4 hours.

[0034] It is known that if the predetermined heating rate is too high, for example, above 10°C / hour, the temperature difference between the inside and outside of the frozen body is large, causing rapid sublimation of internal ice crystals, ultimately leading to cracking or bubbling of the frozen body. Simultaneously, the significant thermal shock may cause denaturation or aggregation of antibody proteins. Conversely, if the predetermined heating rate is too low, for example, below 5°C / hour, it significantly increases the preparation time of the lyophilized reagent balls and slows down the sublimation rate of ice crystals within the lyophilized reagent balls, resulting in fewer pores within the lyophilized reagent balls. Therefore, to address these issues, in some embodiments, the predetermined heating rate satisfies the relationship 5°C / hour ≤ V ≤ 10°C / hour. When the predetermined heating rate is within this range, water vapor within the frozen body can quickly sublimate and escape, forming more pores on the lyophilized reagent balls. This allows the sample to enter the lyophilized reagent balls through the pores after contact with the lyophilized reagent balls, enabling rapid rehydration. Simultaneously, the sublimation rate of ice crystals is not too rapid, avoiding cracking or bubbling of the frozen body. In this embodiment, the value of V can be 5℃ / hour, 6℃ / hour, 7℃ / hour, 8℃ / hour, 9℃ / hour, or 10℃ / hour.

[0035] In some embodiments, the second predetermined temperature T2 satisfies the relationship: -15℃ ≤ T2 ≤ -5℃. This temperature range matches the optimal conditions for ice crystal sublimation, ensuring that sublimation continues without causing the ice crystals to melt or the microspheres to collapse due to excessively high temperatures. The second predetermined temperature T2 can be -15℃, -14℃, -13℃, -12℃, -11℃, -10℃, -9℃, -8℃, -7℃, -6℃, or -5℃.

[0036] In some embodiments, the third predetermined temperature T3 satisfies the relationship: 25℃ ≤ T3 ≤ 30℃. In some embodiments, the second predetermined time A2 satisfies the relationship: 4 hours ≤ A2 ≤ 8 hours. This temperature and duration can completely remove bound water, controlling the residual moisture content to below 3%, meeting the requirements for long-term storage. Simultaneously, the temperature does not exceed 30℃ to avoid antibody denaturation and inactivation due to high temperatures. Furthermore, the reasonable drying time avoids an excessively long overall preparation cycle, reducing the preparation cost of lyophilized reagent balls. In this embodiment, T3 can be 25℃, 26℃, 27℃, 28℃, 29℃, or 30℃. A2 can be 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours.

[0037] In some embodiments, the reaction enhancer includes at least one of polyethylene glycol 4000 and polyethylene glycol 6000.

[0038] Specifically, polyethylene glycol 4000 and polyethylene glycol 6000 can promote the specific binding of antigens and antibodies, thereby shortening the agglutination reaction time and improving the detection response speed. Simultaneously, polyethylene glycol 4000 and polyethylene glycol 6000 can increase the particle size and aggregation degree of immune complexes, significantly improving the sensitivity and absorbance difference of immunoturbidimetric detection, making them suitable for the detection of micro-samples. Furthermore, polyethylene glycol 4000 and polyethylene glycol 6000 can reduce the interference of endogenous substances such as lipoproteins and bilirubin in veterinary serum and plasma, improving detection specificity and repeatability. Of course, polyethylene glycol 8000 and polyethylene glycol 10000 can also be used as reaction enhancers.

[0039] In some embodiments, the surfactant includes at least one of polysorbate 20 and polysorbate 80. In this embodiment, polysorbate 20 and polysorbate 80 can reduce the surface tension of the mixed solution, preventing phenomena such as liquid adhesion, tailing, or stringing when the dispersed mixed solution forms droplets, thus ensuring the sphericity of the microspheres after liquid nitrogen freezing. On the other hand, polysorbate 20 and polysorbate 80 can encapsulate the antibody and its backbone structure, inhibiting antibody molecule aggregation and reducing protein denaturation during freeze-drying. Furthermore, polysorbate 20 and polysorbate 80 can also block hydrophobic sites on the surface of the freeze-dried reagent spheres, reducing non-specific binding of impurities in the sample and lowering background noise. Additionally, Pluronic F68 can also be used as the surfactant.

[0040] In some embodiments, the specific antibody includes at least one of canine C-reactive protein-specific antibody and feline serum amyloid A antibody. Of course, the specific antibody can also be other antibodies, such as canine serum amyloid A-specific antibody, feline C-reactive protein-specific antibody, α1-acid glycoprotein-specific antibody, and heparin-binding protein-specific antibody, etc.

[0041] In some embodiments, the concentration of specific antibodies in the mixed solution is 0.2 mg / mL to 1.5 mg / mL. Specifically, if the antibody concentration is too low, for example, less than 0.2 mg / mL, there are insufficient immunobinding sites between the antigen and antibody, resulting in low detection sensitivity and a narrow linear range. If the antibody concentration is too high, for example, greater than 1.5 mg / mL, this can lead to self-aggregation and non-specific binding between antibodies, resulting in increased background turbidity, increased false positives, and easy collapse of lyophilized reagent spheres. When the antibody concentration is within the above-mentioned range, there are sufficient immunobinding sites between the antigen and antibody, and non-specific binding such as self-aggregation between antibodies is less likely to occur, effectively avoiding the problem of large errors in immunoassay. In this embodiment, the concentration of the specific antibody in the mixed solution can be 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, or 1.5 mg / mL.

[0042] In some embodiments, the mass-volume percentage concentration of the reaction enhancer in the mixed solution is 3%-6%. If the mass-volume percentage concentration of the reaction enhancer in the mixed solution is too low, for example, less than 3%, the agglutination enhancement effect is insufficient, resulting in a weak turbidity signal and poor sensitivity; while if the concentration is too high, for example, greater than 6%, it will induce non-specific antibody agglutination, leading to increased background and narrowing of the linear range. When the concentration is within the above-mentioned range, the agglutination enhancement effect can be maximized while ensuring detection specificity. The mass-volume percentage concentration of the reaction enhancer in the mixed solution can be 3%, 4%, 5%, or 6%.

[0043] In some embodiments, the mass-volume percentage concentration of the surfactant in the mixed solution is 0.001%-0.005%. Specifically, if the mass-volume percentage concentration of the surfactant in the mixed solution is too low, for example, less than 0.001%, the wetting, dispersion, and anti-aggregation effects are insufficient, resulting in poor droplet formation and slow reconstitution of lyophilized reagent beads. If the concentration is too high, for example, greater than 0.005%, it may interfere with the binding of antigen and antibody, inhibit immunoagglutination, and reduce the detection signal. When the above range is met, it can play an interface optimization role without affecting the immune reaction itself. The mass-volume percentage concentration of the surfactant in the mixed solution can be 0.001%, 0.002%, 0.003%, 0.004%, and 0.005%.

[0044] In some embodiments, the step after freeze-drying the frozen body to form lyophilized reagent spheres further includes: screening lyophilized reagent spheres with a diameter conforming to a predetermined diameter R in a dry inert gas environment. Specifically, a dry inert gas environment can isolate moisture and oxygen, preventing the lyophilized reagent spheres from becoming damp or from coming into contact with oxygen, which could lead to antibody oxidation and inactivation. Furthermore, by screening lyophilized reagent spheres with a diameter conforming to the predetermined diameter R, the quality, component content, and reconstitution rate of the finished product are ensured to be consistent, improving the reproducibility of the detection.

[0045] In some embodiments, the predetermined diameter R satisfies the relationship: 0.5 mm ≤ R ≤ 0.8 mm. Specifically, when the predetermined diameter satisfies the above relationship, the selected lyophilized reagent pellets have a small particle size and short pore path, allowing for rapid reconstitution after contact with the sample; simultaneously, the lyophilized reagent pellets within this particle size range exhibit strong structural stability, preventing breakage and fragmentation during transportation or transfer. In this embodiment, the predetermined diameter R can be 0.5 mm, 0.6 mm, 0.7 mm, or 0.8 mm.

[0046] In the screening process of lyophilized reagent beads, some embodiments employ a double-layer screening method, i.e., setting up two sieves: a first sieve with a large aperture and a second sieve with a small aperture. Lyophilized reagent beads larger than a predetermined diameter R cannot pass through the large aperture sieve and are thus screened out. Lyophilized reagent beads smaller than the predetermined diameter R will pass through the small aperture sieve. Ultimately, the lyophilized reagent beads that do not pass through the small aperture sieve are considered to meet the standard.

[0047] In some embodiments, the gas in the dry inert gas environment includes at least one of nitrogen, helium, neon, argon, krypton, or xenon.

[0048] Specifically, nitrogen is low in cost and highly stable, and it does not chemically react with any reagent components such as antibodies, trehalose, hydroxypropyl-β-cyclodextrin, and polyethylene glycol at room temperature. Argon has even higher inertness and does not react with any substances at room temperature. Helium has better thermal conductivity than argon, allowing for rapid temperature transfer. Neon is more stable than argon and helium, does not affect antibodies, and has better optical compatibility. Krypton provides a better gas sealing effect than argon, effectively isolating water vapor and oxygen. Xenon has the strongest inertness, completely preventing antibodies in the lyophilized reagent pellets from contacting oxygen. In practice, the protective gas can be selected based on actual needs when preparing lyophilized reagent pellets.

[0049] To verify the effect of hydroxypropyl-β-cyclodextrin content on the performance of the reagent beads, this application also provides the following specific examples: Example 1: With a trehalose concentration of 3.5% and a hydroxypropyl-β-cyclodextrin concentration of 0.5%, the antibody survival rate was 88.7%. The lyophilized reagent spheres were spherical with uniform pore distribution, showing no obvious shrinkage or breakage, and the finished product qualification rate was 95.2%.

[0050] Example 2: Unlike Example 1, the concentration of hydroxypropyl-β-cyclodextrin was 0.7%, the antibody survival rate was 91.2%, the lyophilized reagent balls were spherical and uniformly distributed with no obvious shrinkage or breakage, and the finished product qualification rate was 97.8%.

[0051] Example 3: Unlike Example 1, the concentration of hydroxypropyl-β-cyclodextrin was 1%, the antibody survival rate was 93.5%, the lyophilized reagent balls were spherical and uniformly distributed with no obvious shrinkage or breakage, and the finished product qualification rate was 98.6%.

[0052] Example 4: Unlike Example 1, the concentration of hydroxypropyl-β-cyclodextrin was 1.3%, the antibody survival rate was 92.8%, the lyophilized reagent balls were spherical and uniformly distributed with no obvious shrinkage or breakage, and the finished product qualification rate was 98.3%.

[0053] Example 5: Unlike Example 1, the concentration of hydroxypropyl-β-cyclodextrin was 1.5%, the antibody survival rate was 92.1%, the lyophilized reagent balls were spherical and uniformly distributed with no obvious shrinkage or breakage, and the finished product qualification rate was 98.0%.

[0054] Example 6: Unlike Example 1, the concentration of hydroxypropyl-β-cyclodextrin was 1.7%, the antibody survival rate was 91.5%, the lyophilized reagent balls were spherical and uniformly distributed with no obvious shrinkage or breakage, and the finished product qualification rate was 97.7%.

[0055] Example 7: Unlike Example 1, the concentration of hydroxypropyl-β-cyclodextrin was 2%, the antibody survival rate was 89.3%, the lyophilized reagent balls were spherical and uniformly distributed with no obvious shrinkage or breakage, and the finished product qualification rate was 95.7%.

[0056] Comparative Example 1: Unlike Example 1, the concentration of hydroxypropyl-β-cyclodextrin was 0.3%, the antibody survival rate was 72.3%, some lyophilized reagent balls showed slight shrinkage and surface irregularity, and there were a few broken balls. The finished product qualification rate was 81.5%.

[0057] Comparative Example 2: Unlike Example 1, the concentration of hydroxypropyl-β-cyclodextrin was 2.5%, the antibody survival rate was 78.5%, the surface of some lyophilized reagent balls was dense, and there were slight flocculent substances after reconstitution. A small number of lyophilized reagent balls had collapse problems, and the finished product qualification rate was 85.3%.

[0058] Comparative Example 3: Unlike Example 1, the concentration of hydroxypropyl-β-cyclodextrin was 3%, the antibody survival rate was 74.1%, most of the lyophilized reagent balls collapsed and adhered, the porous structure was dense, the reconstitution was slow, a small number of broken balls were present, and the finished product qualification rate was 79.8%.

[0059] Based on the above examples and comparative examples, it can be seen that when the concentration of hydroxypropyl-β-cyclodextrin is in the range of 0.5%-2%, the antibody survival rate is above 88%, and the lyophilized reagent balls have regular morphology and high molding qualification rate, indicating that this range can effectively achieve antibody activity protection and microsphere morphology control; when the concentration is below 0.5%, the antibody survival rate decreases significantly (≤79.5%), and the lyophilized reagent balls are prone to shrinkage and breakage; when the concentration is above 2%, the antibody survival rate decreases (≤78.5%), and the lyophilized reagent balls are prone to densification and collapse.

[0060] In addition, to verify the effect of trehalose concentration on the performance of the reagent beads, this application also provides the following specific embodiments: Example 8: The concentration of hydroxypropyl-β-cyclodextrin was 1%, the concentration of trehalose was 2%, the antibody survival rate was 88.2%, the lyophilized reagent spheres were spherical and regular, with no obvious shrinkage or breakage, and the finished product qualification rate was 95.1%.

[0061] Example 9: Unlike Example 8, the concentration of trehalose was 3%, the antibody survival rate was 93.7%, the lyophilized reagent balls were spherical and regular, with no obvious shrinkage or breakage, and the finished product qualification rate was 98.7%.

[0062] Example 10: Unlike Example 8, the concentration of trehalose was 4%, the antibody survival rate was 94.2%, the lyophilized reagent balls were spherical and regular, with no obvious shrinkage or breakage, and the finished product qualification rate was 98.9%.

[0063] Example 11: Unlike Example 8, the concentration of trehalose was 5%, the antibody survival rate was 89.5%, the lyophilized reagent balls were spherical and regular, with no obvious shrinkage or breakage, and the finished product qualification rate was 95.5%.

[0064] Comparative Example 4: Unlike Example 8, the concentration of trehalose was 1%, the antibody survival rate was 70.8%, the structure of the lyophilized reagent balls was loose, some of the lyophilized reagent balls collapsed and shrank and deformed, and the finished product qualification rate was 78.6%.

[0065] Comparative Example 5: Unlike Example 8, the concentration of trehalose was 1.5%, the antibody survival rate was 79.5%, some lyophilized reagent balls showed slight shrinkage and deformation, a small number of lyophilized reagent balls were crushed, and the finished product qualification rate was 87.2%.

[0066] Comparative Example 6: Unlike Example 8, the concentration of trehalose was 6%, the antibody survival rate was 77.3%, the viscosity of the mixed solution was too high, the size of the dispersed droplets was inconsistent, and the prepared lyophilized reagent balls were dense and slow to reconstitute, with a finished product qualification rate of 84.9%.

[0067] Comparative Example 7: Unlike Example 8, the concentration of trehalose was 7%, the antibody survival rate was 73.6%, the viscosity of the mixed solution was too high, the dispersed droplets were stringy, resulting in different droplet sizes, and some of the prepared lyophilized reagent balls were dense and slow to reconstitute, with flocculent matter after reconstitution. The finished product qualification rate was 78.3%.

[0068] As can be seen from the above examples and comparative examples, when the trehalose concentration is in the range of 2%-5%, the antibody survival rate is above 88%, the microspheres have regular morphology and good resolubility; when the concentration is below 2%, the antibody survival rate decreases significantly (≤79.5%), the microsphere skeleton is loose and easy to collapse; when the concentration is above 5%, the antibody survival rate decreases (≤77.3%), the reaction solution has poor formation and is easy to stick together.

[0069] In addition, to illustrate the effect of trehalose and hydroxypropyl-β-cyclodextrin concentrations on the reconstitution efficiency of lyophilized reagent beads, this application also provides the following specific examples: Example 12: The concentration of trehalose was 2%, the concentration of hydroxypropyl-β-cyclodextrin was 0.5%, the reconstitution time was 18 seconds, the dissolution was uniform and there were no lumps, and the lyophilized reagent spheres were regular in shape and not broken.

[0070] Example 13 With a trehalose concentration of 3% and a hydroxypropyl-β-cyclodextrin concentration of 0.8%, the reconstitution time is 13 seconds, resulting in rapid dissolution without clumping. The lyophilized reagent spheres exhibit a uniform porous structure and moderate mechanical strength.

[0071] Example 14 The trehalose concentration was 3.5%, the hydroxypropyl-β-cyclodextrin concentration was 1.2%, the reconstitution time was 12 seconds, and it dissolved rapidly and completely without clumping or turbidity. The lyophilized reagent spheres had regular morphology, and the system was homogeneous after reconstitution.

[0072] Example 15 The trehalose concentration was 4.5%, the hydroxypropyl-β-cyclodextrin concentration was 1.8%, the reconstitution time was 14 seconds, the dissolution was uniform and there was no clumping, the lyophilized reagent balls had good mechanical strength and no dust was generated.

[0073] Example 16 With a trehalose concentration of 5% and a hydroxypropyl-β-cyclodextrin concentration of 2%, the reconstitution time was 17 seconds. The solution was uniformly dissolved without clumping, and the lyophilized reagent spheres were regular in shape and without breakage.

[0074] Comparative Example 8 The concentration of trehalose was 1.5%, the concentration of hydroxypropyl-β-cyclodextrin was 0.3%, the reconstitution time was 38 seconds, slight clumping occurred during the reconstitution process, the dissolution was slow, and the skeleton of the lyophilized reagent spheres was loose and partially broken.

[0075] Comparative Example 9 With a trehalose concentration of 6% and a hydroxypropyl-β-cyclodextrin concentration of 2.5%, the reconstitution time was 42 seconds. The reconstitution was slow, and local insoluble flocculent matter appeared. The lyophilized reagent spheres had a dense structure and closed pores.

[0076] As shown in the above examples and comparative examples, when the trehalose concentration is between 2% and 5% and the hydroxypropyl-β-cyclodextrin concentration is between 0.5% and 2%, the reconstitution time of the lyophilized reagent beads is between 12 and 18 seconds, achieving rapid reconstitution with uniformity and no clumping, while also maintaining the regularity of the microsphere morphology and mechanical strength. When both concentrations are below the specified range, the reconstitution time is significantly prolonged (≥38 seconds), and clumping and microsphere breakage are likely to occur. When both concentrations are above the specified range, the reconstitution time is also prolonged (≥42 seconds), and the lyophilized reagent beads have a dense structure and closed pores, making rapid reconstitution impossible.

[0077] Furthermore, taking canine CRP detection as an example, this application provides the following specific examples of preparing lyophilized reagent beads and conducting the detection: (1) Take anti-canine CRP antibody with a concentration of 0.8 mg / mL, 4% PEG8000, 3.5% trehalose, 1.2% hydroxypropyl-β-cyclodextrin, and 0.03% Pronic F68, and adjust the volume to 100 mL with pH 7.4 phosphate buffer. Stir well, let stand to degas, and set aside for later use.

[0078] (2) The prepared mixed solution is dripped into liquid nitrogen at -196℃ through a syringe dripper at a dripping speed of 5 drops / minute to form uniform droplets with a droplet diameter of 1.0-1.5 mm. The droplets are vitrified and frozen instantly upon contact with the liquid nitrogen to form a frozen body with a complete spherical skeleton, thus avoiding the damage of the antibody structure by large ice crystals.

[0079] (3) Transfer the frozen body into a vacuum freeze-drying chamber, set the temperature to -40℃ and the vacuum degree to 8 Pa, and maintain for 3 hours; this parameter ensures that the free ice crystals inside the frozen body sublimate smoothly, fixes the spherical skeleton, and protects the antibody activity. Slowly raise the temperature to -10℃ at a rate of 8℃ per hour and maintain for 12 hours; this heating rate can achieve simultaneous sublimation of ice crystals inside and outside the frozen body, avoid residual moisture caused by surface sealing, and balance drying efficiency and antibody protection. Continue to raise the temperature to 25℃ and maintain for 6 hours; completely remove the bound water in the system, control the residual moisture of the freeze-dried reagent balls to below 3%, and improve storage stability.

[0080] (4) Take out the lyophilized reagent balls under the protection of high-purity dry nitrogen, and screen them using a stacked standard vibrating sieve. Select the lyophilized reagent balls with a particle size of 0.5mm-0.8mm as the finished product; collect and discard the products with other particle sizes separately.

[0081] The purpose of using a phosphate buffer solution with a pH of 7.4 is to maintain a stable pH of 7.4 in the reaction solution system. This pH value matches the isoelectric point of canine CRP antibodies, minimizing conformational changes and denaturation caused by pH fluctuations, thus ensuring the antibody's immunobinding activity. Simultaneously, it provides a stable ionic environment, inhibiting antibody molecule self-aggregation, promoting the enhanced reaction of PEG8000, and improving immunoagglutination efficiency. Furthermore, the buffer system itself is chemically stable, not decomposing during liquid nitrogen deep freezing and vacuum lyophilization, and does not react with other components in the system, ensuring the stability of the lyophilized reagent beads. After reconstitution of the lyophilized reagent beads, the buffer solution can quickly adapt to the pH environment of the test sample, ensuring smooth immunoturbidimetric reactions and improving detection repeatability. Sodium dihydrogen phosphate-disodium hydrogen phosphate buffer or potassium dihydrogen phosphate-disodium hydrogen phosphate buffer are preferred options, and can be used alone or in combination. Sodium dihydrogen phosphate-disodium hydrogen phosphate buffer is the most commonly used. It has good compatibility with the components of this reaction system, is easy to prepare, has low cost, and can stably maintain a buffer range of pH 7.4 without adversely affecting antibody activity and lyophilization.

[0082] (5) The prepared lyophilized reagent spheres have a regular spherical porous structure, a reconstitution time of 12 seconds, an antibody survival rate of 94.3% (antibody activity was detected by ELISA, and was the ratio of antibody activity before lyophilization), and a bulk density of 0.25 g / cm³. 3The linear range of CRP in canine samples was 0.1-100 mg / L, with a CV value of <5%. The detection sensitivity and specificity met the design requirements. After long-term storage (sealed storage at 25℃ for 6 months), the antibody activity retention rate was still above 88%, and the reconstitution time was not significantly prolonged.

[0083] On the other hand, this application also provides a lyophilized reagent ball for immunoassay, which is prepared by the above-described preparation method for lyophilized reagent balls for immunoassay. Since the technical effects of the lyophilized reagent ball for immunoassay have already been described in the preparation method for lyophilized reagent balls for immunoassay, they will not be repeated here.

[0084] For ease of description, spatial relative terms such as "above," "on top of," "on the upper surface of," "above," etc., are used herein to describe the spatial positional relationship of a device or feature as shown in the figures to other devices or features. It should be understood that spatial relative terms are intended to encompass different orientations in use or operation beyond the orientation of the device as described in the figures. For example, if the device in the figures were inverted, a device described as "above" or "on top of" other devices or structures would subsequently be positioned as "below" or "under" other devices or structures. Thus, the exemplary term "above" can include both "above" and "below." The device may also be positioned in other different ways (rotated 90 degrees or in other orientations), and the spatial relative descriptions used herein will be interpreted accordingly.

[0085] Furthermore, it should be noted that the use of terms such as "first" and "second" to define components is merely for the purpose of distinguishing the corresponding components. Unless otherwise stated, the above terms have no special meaning and therefore cannot be construed as limiting the scope of protection of this application.

[0086] The above are merely preferred embodiments of this application and are not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for preparing lyophilized reagent beads for immunoassay, characterized in that, include: The specific antibody, reaction enhancer, lyophilization excipient, and surfactant are mixed into a mixed solution; The mixed solution is dispersed into multiple droplets of equal volume, and each droplet is frozen to form a frozen body; The frozen body was freeze-dried to form freeze-dried reagent spheres; The freeze-drying excipients include at least trehalose and hydroxypropyl-β-cyclodextrin.

2. The method for preparing lyophilized reagent beads for immunoassay according to claim 1, characterized in that, The trehalose concentration in the freeze-drying excipient is 2%-5% by mass volume; and / or, The hydroxypropyl-β-cyclodextrin concentration in the lyophilized excipient is 0.5%-2% by mass volume.

3. The method for preparing lyophilized reagent beads for immunoassay according to claim 1, characterized in that, The step of dispersing the mixed solution into multiple droplets of equal volume and freezing each droplet to form a frozen body includes: The mixed solution is used to form multiple droplets of equal volume using a quantitative titration device; The liquid droplets are added to liquid nitrogen to prepare the frozen body.

4. The method for preparing lyophilized reagent balls for immunoassay according to any one of claims 1 to 3, characterized in that, The step of freeze-drying the frozen body to form freeze-dried reagent spheres includes: Under a predetermined vacuum level P, the frozen body is frozen for a first predetermined time A1 within a first predetermined temperature T1. The temperature is increased to a second predetermined temperature T2 at a predetermined heating rate V, so that the ice crystals in the frozen body sublimate. The frozen body is heated to a third predetermined temperature T3 at the predetermined heating rate V, and then dried for a second predetermined time A2 to form the lyophilized reagent ball.

5. The method for preparing lyophilized reagent beads for immunoassay according to claim 4, characterized in that, The predetermined vacuum level P satisfies the relationship: P < 10 Pa; and / or, The first predetermined temperature T1 satisfies the following relationship: -45℃≤T1≤-35℃; and / or, The first predetermined time A1 satisfies the following relationship: 2 hours ≤ A1 ≤ 4 hours; and / or, The predetermined heating rate satisfies the relationship 5℃ / hour ≤ V ≤ 10℃ / hour; and / or, The second predetermined temperature T2 satisfies the following relationship: -15℃ ≤ T2 ≤ -5℃; and / or, The third predetermined temperature T3 satisfies the following relationship: 25℃≤T3≤30℃; and / or, The second predetermined time A2 satisfies the relationship 4 hours ≤ A2 ≤ 8 hours.

6. The method for preparing lyophilized reagent balls for immunoassay according to any one of claims 1 to 3, characterized in that, The reaction enhancer includes at least one of polyethylene glycol 4000 and polyethylene glycol 6000; and / or, The surfactant includes at least one of polysorbate 20 and polysorbate 80; and / or, The specific antibody includes at least one of canine C-reactive protein specific antibody and feline serum amyloid A antibody.

7. The method for preparing lyophilized reagent balls for immunoassay according to any one of claims 1 to 3, characterized in that, The concentration of the specific antibody in the mixed solution is 0.2 mg / mL to 1.5 mg / mL; and / or, In the mixed solution, the mass-volume percentage concentration of the reaction enhancer is 3%-6%; and / or, In the mixed solution, the concentration of the surfactant by mass volume percentage is 0.001%-0.005%.

8. The method for preparing lyophilized reagent balls for immunoassay according to any one of claims 1 to 3, characterized in that, The step following freeze-drying the frozen body to form freeze-dried reagent spheres also includes: In a dry, inert gas environment, the lyophilized reagent spheres with a diameter conforming to a predetermined diameter R are selected.

9. The method for preparing lyophilized reagent beads for immunoassay according to claim 8, characterized in that, The predetermined diameter R satisfies the following relationship: 0.5mm ≤ R ≤ 0.8mm; and / or, The gas in the dry inert gas environment includes at least one of nitrogen, helium, neon, argon, krypton, or xenon.

10. A lyophilized reagent ball for immunoassay, characterized in that, The lyophilized reagent balls for immunoassay are prepared using the method described in any one of claims 1 to 9.

Images