A freeze-dried microsphere for luminescence immunoassay and a preparation method thereof

Abstract

The application provides a freeze-dried microsphere for luminescence immunoassay and a preparation method thereof. The preparation method comprises the following steps: mixing prepared chemiluminescence immunoassay reagents and a protective agent in a volume ratio of 1:1.5, dropping into liquid nitrogen to form microspheres, and performing vacuum freeze drying on the microspheres to form freeze-dried microspheres; and the protective agent comprises trehalose, mannitol, soybean oil, 1,2 hexanediol and octaglycerol. The preparation method of the freeze-dried microsphere has high microsphere hardness and is not prone to cracking, especially has good reconstitution effect, and the active ingredients contained in the reagent are stable during use, and have no any influence on the accuracy and sensitivity of the detection itself.

Description

Technical Field

[0001] This invention relates to the field of luminescent immunoassay, and more specifically, to a lyophilized microsphere for luminescent immunoassay and its preparation method. Background Technology

[0002] In recent years, chemiluminescence immunoassay technology has developed rapidly, and its applications in medicine, life sciences, and other fields have been continuously expanding. Immunological detection primarily utilizes the specific reaction between antigens and antibodies for detection. Because it can utilize isotopes, enzymes, chemiluminescent substances, etc., to amplify and display the detection signal, it is often used to detect trace substances such as proteins and hormones. Chemiluminescence immunoassay is a novel labeled immunoassay technique that combines a chemiluminescent substrate with an immune reaction to detect trace amounts of antigens or antibodies. The principle of the chemiluminescence reaction is to label an antigen (or antibody) with a luminescent substance or enzyme, excite the luminescent substance to emit light through substrate oxidation, and then detect it using a chemiluminescence analyzer.

[0003] Currently, chemiluminescence product reagent kits are all packaged in liquid form. Since the core raw materials (antigens or antibodies) used in chemiluminescence products are bioactive substances, they easily lose their bioactivity at room temperature. Therefore, all liquid reagent kits require cold chain transportation and low-temperature storage, which is quite troublesome. Although existing technologies use lyophilized microspheres to replace liquid reagents to solve the transportation and low-temperature storage problems, the lyophilized microspheres generally have problems such as harsh storage conditions, easy cracking and low hardness, unstable active ingredients in the reagents, and poor reconstitution. These issues affect the accuracy and sensitivity of the detection. Summary of the Invention

[0004] The purpose of this invention is to provide a lyophilized microsphere for luminescent immunoassay and its preparation method. The lyophilized microspheres prepared by the method of this invention are easy to store, have high hardness and are not easy to crack, and have good reconstitution effect. Moreover, the active ingredients contained in the reagent are stable during use and have no impact on the accuracy and sensitivity of the detection itself.

[0005] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0006] This invention provides a method for preparing lyophilized microspheres for luminescent immunoassay, comprising the following steps:

[0007] The prepared chemiluminescent immunoassay reagent and the protective agent were mixed at a volume ratio of 1:1.5, and then added to liquid nitrogen to form microspheres. The microspheres were then freeze-dried under vacuum to form lyophilized microspheres.

[0008] The protective agents include: trehalose, mannitol, soybean oil, 1,2-hexanediol, and caprylyl glycol.

[0009] Preferably, as a further feasible option, the following components are included by weight: 1-5 parts trehalose, 2-6 parts mannitol, 0.6-1.5 parts soybean oil, 0.8-1.2 parts 1,2-hexanediol, and 0.1-0.5 parts octyl glycol.

[0010] Preferably, as a further feasible option, the ingredients are, by weight, 2-4 parts trehalose, 3-5 parts mannitol, 0.8-1.3 parts soybean oil, 0.9-1.1 parts 1,2-hexanediol, and 0.2-0.4 parts caprylyl glycol.

[0011] Preferably, as a further feasible option, the mixture comprises 3 parts trehalose, 4 parts mannitol, 1.1 parts soybean oil, 1.0 part 1,2-hexanediol, and 0.3 parts caprylyl glycol.

[0012] In the microsphere freeze-drying process of this invention, the stability of the active ingredients is improved by mixing mannitol, trehalose, soybean oil, 1,2-hexanediol, and caprylyl glycol with chemiluminescent immunoassay reagents. In particular, the simultaneous addition of 1,2-hexanediol and caprylyl glycol synergistically enhances the stabilizing effect of the active ingredients, reduces the influence of the surrounding environment on the microspheres, and especially avoids the aggregation of some components that are prone to combining with the active ingredients and causing deterioration on the surface of the microspheres, thus providing comprehensive protection for the microspheres. Moreover, the addition of the above-mentioned components does not affect the detection function of the reagents themselves. In particular, the addition of a small amount of soybean oil and the combination with trehalose have a synergistic effect, forming a non-hygroscopic protective layer on the surface of the microspheres, which provides low-temperature protection and dehydration protection, and improves the shape of the microspheres themselves.

[0013] Of course, through continuous practice, it was found that the specific components of the compound and the controlled dosage of each component were optimized. In particular, the specific combination of mannitol with 1,2-hexanediol and caprylyl glycol, and the specific combination of soybean oil and trehalose, showed that other components could not produce such a good synergistic effect. Regarding the dosage, it was also continuously explored based on the stability of the microspheres during specific operations. The dosage of 1,2-hexanediol and caprylyl glycol was much less than that of mannitol, because their addition mainly serves to protect the active ingredients. If the dosage is too high, it will affect the effectiveness of the active ingredients themselves. The dosage of soybean oil should be kept as low as possible, just enough to provide moisture resistance. If the dosage is too high, it will be difficult to degrade and will also affect the normal use of the microspheres.

[0014] Preferably, as a further feasible option, sorbic acid and oxalic acid are added before freeze-drying with liquid nitrogen.

[0015] Preferably, as a further feasible option, the sorbic acid content is 0.2-0.9 parts by weight, and the oxalic acid content is 0.09-0.1 parts by weight.

[0016] To improve the resolvability of freeze-dried microspheres, this invention adds sorbic acid and oxalic acid along with a protective agent. By synergistically combining the two and controlling their dosage within a suitable range, the solubility of the microspheres can be accelerated, allowing them to quickly disperse into a uniform system. This not only results in rapid dissolution but also eliminates any solid residue after dissolution, ensuring uniform dispersion and ease of use. This not only improves work efficiency but also enhances detection efficiency.

[0017] Preferably, as a further feasible option, the freeze-drying process is carried out in multiple stages, wherein the first freeze-drying stage lasts for 1-10 hours and the temperature is -40 to -55°C.

[0018] Preferably, as a further feasible option, the freeze-drying process is carried out in three stages: the second freeze-drying stage lasts for 2-8 hours at a temperature of -30 to -35°C; the third freeze-drying stage is a drying stage lasting for 4-7 hours at a temperature of 20-40°C.

[0019] Preferably, as a further feasible option, the freeze-drying process is carried out under a vacuum of 70-120 ubar.

[0020] To improve the stability of the microspheres, this invention implements programmed control over the entire freeze-drying process, dividing it into multiple stages. In each stage, the freeze-drying temperature and time are adjusted. By precisely controlling the temperature and time, the active ingredients in the microspheres are not easily lost, they are not easily cracked, and they have good formability during the freeze-drying process.

[0021] In summary, the freeze-dried microspheres prepared by the above method have good shape retention and better performance in subsequent use.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] (1) The microsphere freeze-drying process of the present invention allows for more precise control of the reagents and operating parameters, making the obtained freeze-dried microspheres easy to preserve and with high hardness that are not prone to cracking.

[0024] (2) The microspheres prepared by the freeze-dried microsphere preparation method of the present invention can be quickly dispersed into a uniform system, which is convenient to use, improves work efficiency, and also improves detection efficiency.

[0025] (3) The microspheres prepared by the freeze-drying process of the present invention are not easily affected by oxygen even when stored in an air atmosphere, thus affecting the quality of the microspheres. The microspheres themselves have good stability and excellent moisture resistance. Detailed Implementation

[0026] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0027] Example 1

[0028] The lyophilized preparation method of microspheres for chemiluminescent immunoassay is carried out according to the following steps:

[0029] (1) Pre-preparation of protective agent: 1g trehalose, 6g mannitol, 0.6g soybean oil, 1.2g 1,2-hexanediol, 0.1g caprylyl glycol, 0.2g sorbic acid, 0.1g oxalic acid and 1000ml purified water are stirred and dissolved, and the resulting solution is filtered through a filter membrane;

[0030] (2) Add the prepared luminescent immunoassay reagent to the protective agent in step (1) above. The volume ratio of the luminescent immunoassay reagent to the effective component of the protective agent is 1:1.5.

[0031] (3) After mixing, liquid nitrogen is added to form microspheres, and the microspheres are freeze-dried under vacuum to form freeze-dried microspheres;

[0032] (4) The freeze-drying process is carried out in three stages. The first freeze-drying stage lasts for 1 hour at a temperature of -55℃, the second freeze-drying stage lasts for 8 hours at a temperature of -30℃, and the third freeze-drying stage is the drying stage, lasts for 7 hours at a temperature of 20℃, and the vacuum degree is between 70-120 ubar.

[0033] (5) The freeze-dried microspheres obtained in the above steps are sealed and stored, and can be directly used for chemiluminescence immunoassay.

[0034] Example 2

[0035] The lyophilized preparation method of microspheres for chemiluminescent immunoassay is carried out according to the following steps:

[0036] (1) Pre-preparation of protective agent: 5g trehalose, 2g mannitol, 1.5g soybean oil, 0.8g 1,2-hexanediol, 0.5g caprylyl glycol, 0.9g sorbic acid, 0.09g oxalic acid and 1000ml purified water are stirred and dissolved, and the resulting solution is filtered through a filter membrane;

[0037] (2) Add the prepared luminescent immunoassay reagent to the protective agent in step (1) above. The volume ratio of the luminescent immunoassay reagent to the effective component of the protective agent is 1:1.5.

[0038] (3) After mixing, liquid nitrogen is added to form microspheres, and the microspheres are freeze-dried under vacuum to form freeze-dried microspheres;

[0039] (4) The freeze-drying process is carried out in three stages. The first freeze-drying stage lasts for 10 hours at a temperature of -40°C. The second freeze-drying stage lasts for 2 hours at a temperature of -35°C. The third freeze-drying stage is the drying stage, lasts for 4 hours at a temperature of 40°C, and the vacuum degree is between 70-120 ubar.

[0040] (5) The freeze-dried microspheres obtained in the above steps are sealed and stored, and can be directly used for chemiluminescence immunoassay.

[0041] Example 3

[0042] The lyophilized preparation method of microspheres for chemiluminescent immunoassay is carried out according to the following steps:

[0043] (1) Pre-preparation of protective agent: 2g trehalose, 5g mannitol, 0.8g soybean oil, 1.1g 1,2-hexanediol, 0.2g caprylyl glycol, 0.9g sorbic acid, 0.09g oxalic acid and 1000ml purified water are stirred and dissolved, and the resulting solution is filtered through a filter membrane;

[0044] (2) Add the prepared luminescent immunoassay reagent to the protective agent in step (1) above. The volume ratio of the luminescent immunoassay reagent to the effective component of the protective agent is 1:1.5.

[0045] (3) After mixing, liquid nitrogen is added to form microspheres, and the microspheres are freeze-dried under vacuum to form freeze-dried microspheres;

[0046] (4) The freeze-drying process is carried out in three stages. The first freeze-drying stage lasts for 7 hours at a temperature of -45°C, the second freeze-drying stage lasts for 6 hours at a temperature of -32°C, and the third freeze-drying stage is the drying stage, lasts for 5 hours at a temperature of 30°C, and the vacuum degree is between 70-120 ubar.

[0047] (5) The freeze-dried microspheres obtained in the above steps are sealed and stored, and can be directly used for chemiluminescence immunoassay.

[0048] Example 4

[0049] The specific operating steps are the same as in Example 3, except that: 4g of trehalose, 3g of mannitol, 1.3g of soybean oil, 0.9g of 1,2-hexanediol, 0.4g of caprylyl glycol, 0.7g of sorbic acid, 0.095g of oxalic acid, and 1000ml of purified water are stirred and dissolved.

[0050] Example 5

[0051] The specific operating steps are the same as in Example 3, except that: 3g of trehalose, 4g of mannitol, 1.1g of soybean oil, 1.0g of 1,2-hexanediol, 0.3g of caprylyl glycol, 0.7g of sorbic acid, 0.095g of oxalic acid, and 1000ml of purified water are stirred and dissolved.

[0052] Example 6

[0053] The specific operating steps are the same as in Example 3, except that sorbic acid and oxalic acid are not added.

[0054] Example 7

[0055] The specific operating steps are the same as in Example 3, except that 1g of caprylyl glycol is used.

[0056] Example 8

[0057] The specific operating steps are the same as in Example 3, except that 1.5g of hexanediol is used.

[0058] Comparative Example 1

[0059] The specific operating steps are the same as in Example 3, except that hexanediol is replaced with ethylene glycol.

[0060] Comparative Example 2

[0061] The specific operating steps are the same as in Example 3, except that soybean oil is not added.

[0062] Example 9

[0063] The specific operating steps are the same as in Example 3, except that the freeze-drying process is divided into two stages. The first freeze-drying stage lasts for 7 hours at a temperature of -45°C, and the second freeze-drying stage lasts for 5 hours at a temperature of 30°C with a vacuum degree between 50-150 ubar.

[0064] Experimental Example 1

[0065] The stability of the microspheres in the above embodiments and comparative examples was tested, and the specific results are as follows: the moisture absorption rate of the microspheres after being placed in different ambient humidity environments for 24 hours, and the change in their appearance after being exposed to different temperature ambient environments for 48 hours, were used to demonstrate their stability. The dry environment refers to an ambient humidity of less than 3%.

[0066] Table 1 Experimental Results

[0067]

[0068] As can be seen from the data in Table 1 above, when the microspheres prepared in each embodiment and comparative example are exposed to a humid air environment, although their moisture absorption rate tends to increase with the increase of ambient humidity, the stability of the microspheres is also different because the operating conditions of each embodiment and comparative example are different during the entire freeze-drying process. From the data on moisture absorption rate, the optimal process is Example 5. In Example 5, the added stabilizing components and freeze-drying operating conditions are controlled to be optimal, so its moisture absorption rate and the trend of increase in moisture absorption rate are both low. This indicates that the microspheres of this embodiment can still achieve good stability and moisture resistance even when exposed to a humid air environment. Compared with Example 5, although the operating conditions of Examples 1-4 are slightly different, their stability is still relatively good. Starting from Example 6, since the added sorbic acid and oxalic acid are mainly to increase the resolubility, when these two substances are not added, the shape will not be good, the surface will be relatively rough, and the subsequent dissolution will also affect the dissolution efficiency. After dissolution, a solution with a relatively uniform texture cannot be formed. The data from Examples 7 and 8 show that the addition of alcohols also has a certain dosage relationship. If the amount of octyl glycol and hexanediol is too large, it will affect the compatibility with mannitol, resulting in poor stability of the active ingredients and making them more susceptible to environmental influences, leading to a rapid increase in moisture absorption. Furthermore, the data from Comparative Example 2 shows that the absence of soybean oil also affects the moisture resistance of the microspheres and the final stability of the microspheres. It is evident that although the amount of soybean oil added is relatively large, its addition allows for a good complexation with other substances, ensuring moisture resistance; however, without its addition, the moisture absorption rate increases rapidly. In Comparative Example 1, changing the type of alcohol did not yield the expected results. Therefore, a fixed combination and specific dosage ratio are crucial for ensuring the stability of the microspheres in this invention. Finally, in Example 9, the freeze-drying process was not controlled under optimal conditions, which also affected the final moisture absorption rate.

[0069] As can be seen from the changes in the appearance of the microspheres under different temperature conditions in the table, the best embodiment is Example 5. Other embodiments, starting from Example 6, are affected to varying degrees due to the poor stability of the microspheres.

[0070] In summary, as can be seen from the data in Table 1 above, the freeze-drying preparation method of the present invention requires that all operating conditions be controlled within the optimal range in order to achieve good results.

[0071] Although the invention has been illustrated and described with reference to specific embodiments, it should be understood that many other changes and modifications can be made without departing from the spirit and scope of the invention. Therefore, it is intended that all such changes and modifications falling within the scope of the invention be included in the appended claims.

Claims

1. A method for preparing lyophilized microspheres for luminescent immunoassay, characterized in that, Includes the following steps: The prepared chemiluminescent immunoassay reagent and the protective agent were mixed at a volume ratio of 1:1.5, and then added to liquid nitrogen to form microspheres. The microspheres were then freeze-dried under vacuum to form lyophilized microspheres. The protective agents include: trehalose, mannitol, soybean oil, 1,2-hexanediol, and caprylyl glycol; By weight, it contains 1-5 parts trehalose, 2-6 parts mannitol, 0.6-1.5 parts soybean oil, 0.8-1.2 parts 1,2-hexanediol, and 0.1-0.5 parts caprylyl glycol.

2. The preparation method according to claim 1, characterized in that, By mass fraction, it contains 2-4 parts trehalose, 3-5 parts mannitol, 0.8-1.3 parts soybean oil, 0.9-1.1 parts 1,2-hexanediol, and 0.2-0.4 parts caprylyl glycol.

3. The preparation method according to claim 1, characterized in that, By weight, the composition is as follows: trehalose 3 parts, mannitol 4 parts, soybean oil 1.1 parts, 1,2-hexanediol 1.0 part and caprylyl glycol 0.3 parts.

4. The preparation method according to claim 1, characterized in that, Sorbic acid and oxalic acid are added before freeze-drying with liquid nitrogen.

5. The preparation method according to claim 4, characterized in that, By mass fraction, sorbic acid is 0.2-0.9 parts and oxalic acid is 0.09-0.1 parts.

6. The preparation method according to any one of claims 1-5, characterized in that, The freeze-drying process is carried out in multiple stages, with the first freeze-drying stage lasting 1-10 hours at a temperature of -40 to -55°C.

7. The preparation method according to claim 6, characterized in that, The freeze-drying process is carried out in three stages. The second freeze-drying stage lasts for 2-8 hours at a temperature of -30 to -35°C. The third freeze-drying stage is the drying stage, which lasts for 4-7 hours at a temperature of 20-40°C.

8. The preparation method according to claim 6, characterized in that, The freeze-drying process is carried out under a vacuum of 70-120 ubar.

9. The freeze-dried microspheres obtained by the preparation method according to any one of claims 1-8.

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