CN116832720A - Freeze-dried microsphere for luminous immunodetection and preparation method thereof - Google Patents
Freeze-dried microsphere for luminous immunodetection and preparation method thereof Download PDFInfo
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- 239000004005 microsphere Substances 0.000 title claims abstract description 72
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 238000000034 method Methods 0.000 claims abstract description 33
- 235000012424 soybean oil Nutrition 0.000 claims abstract description 25
- 239000003549 soybean oil Substances 0.000 claims abstract description 25
- 229940015975 1,2-hexanediol Drugs 0.000 claims abstract description 19
- FHKSXSQHXQEMOK-UHFFFAOYSA-N hexane-1,2-diol Chemical compound CCCCC(O)CO FHKSXSQHXQEMOK-UHFFFAOYSA-N 0.000 claims abstract description 19
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims abstract description 18
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims abstract description 18
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims abstract description 18
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims abstract description 17
- 229930195725 Mannitol Natural products 0.000 claims abstract description 17
- 235000010355 mannitol Nutrition 0.000 claims abstract description 17
- 239000000594 mannitol Substances 0.000 claims abstract description 17
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000003223 protective agent Substances 0.000 claims abstract description 16
- OEIJHBUUFURJLI-UHFFFAOYSA-N octane-1,8-diol Chemical compound OCCCCCCCCO OEIJHBUUFURJLI-UHFFFAOYSA-N 0.000 claims abstract description 14
- 238000003018 immunoassay Methods 0.000 claims abstract description 10
- 239000007788 liquid Substances 0.000 claims abstract description 10
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 8
- 238000004020 luminiscence type Methods 0.000 claims abstract description 7
- 239000000203 mixture Substances 0.000 claims abstract description 6
- 238000009777 vacuum freeze-drying Methods 0.000 claims abstract description 6
- 238000002156 mixing Methods 0.000 claims abstract description 3
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 claims description 39
- 238000004108 freeze drying Methods 0.000 claims description 38
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 claims description 13
- 235000006408 oxalic acid Nutrition 0.000 claims description 13
- 229940075582 sorbic acid Drugs 0.000 claims description 13
- 235000010199 sorbic acid Nutrition 0.000 claims description 13
- 239000004334 sorbic acid Substances 0.000 claims description 13
- 238000012792 lyophilization process Methods 0.000 claims description 4
- 239000004480 active ingredient Substances 0.000 abstract description 13
- 230000000694 effects Effects 0.000 abstract description 13
- 238000001514 detection method Methods 0.000 abstract description 9
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 7
- 238000004090 dissolution Methods 0.000 abstract description 7
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 238000005336 cracking Methods 0.000 abstract 1
- KBPLFHHGFOOTCA-UHFFFAOYSA-N 1-Octanol Chemical compound CCCCCCCCO KBPLFHHGFOOTCA-UHFFFAOYSA-N 0.000 description 12
- 238000010521 absorption reaction Methods 0.000 description 8
- 230000000052 comparative effect Effects 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- 239000008213 purified water Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 239000000427 antigen Substances 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- ACCCMOQWYVYDOT-UHFFFAOYSA-N hexane-1,1-diol Chemical compound CCCCCC(O)O ACCCMOQWYVYDOT-UHFFFAOYSA-N 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 230000002195 synergetic effect Effects 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- GLZWNFNQMJAZGY-UHFFFAOYSA-N octaethylene glycol Chemical compound OCCOCCOCCOCCOCCOCCOCCOCCO GLZWNFNQMJAZGY-UHFFFAOYSA-N 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 206010017472 Fumbling Diseases 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000036046 immunoreaction Effects 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000002893 slag Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
- B01J13/04—Making microcapsules or microballoons by physical processes, e.g. drying, spraying
-
- F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
- F26—DRYING
- F26B—DRYING SOLID MATERIALS OR OBJECTS BY REMOVING LIQUID THEREFROM
- F26B5/00—Drying solid materials or objects by processes not involving the application of heat
- F26B5/04—Drying solid materials or objects by processes not involving the application of heat by evaporation or sublimation of moisture under reduced pressure, e.g. in a vacuum
- F26B5/06—Drying solid materials or objects by processes not involving the application of heat by evaporation or sublimation of moisture under reduced pressure, e.g. in a vacuum the process involving freezing
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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Abstract
The invention provides a freeze-dried microsphere for luminescence immunoassay and a preparation method thereof, wherein the preparation method of the freeze-dried microsphere comprises the following steps: mixing the prepared chemiluminescent immunoreagent with a protective agent in a volume ratio of 1:1.5, dripping liquid nitrogen into the mixture to form microspheres, and performing vacuum freeze drying on the microspheres to form freeze-dried microspheres; wherein the protective agent comprises: trehalose, mannitol, soybean oil, 1,2 hexanediol and octanediol. The preparation method of the freeze-dried microsphere has the advantages of high hardness, difficult cracking, good re-dissolution effect, stable active ingredients contained in the reagent in the using process, and no influence on the accuracy and sensitivity of the detection.
Description
Technical Field
The invention relates to the field of luminous immunodetection application, in particular to a freeze-dried microsphere for luminous immunodetection and a preparation method thereof.
Background
In recent years, chemiluminescent immunoassay technology has been rapidly developed, and applications in the fields of medicine, life science and the like have been continuously expanded. Immunological detection is a method for detecting an antigen and an antibody by using a specific reaction, and is often used for detecting trace substances such as proteins and hormones because detection signals can be amplified and displayed by using isotopes, enzymes, chemiluminescent substances, and the like. Chemiluminescent immunoassay is a novel labeled immunoassay technique for detecting minute amounts of antigen or antibody by combining chemiluminescent substrate with an immunoreaction. The principle of the chemiluminescent reaction is to label a luminescent substance or enzyme on an antigen (or antibody), excite the luminescent substance to emit light by oxidizing a substrate, and then detect the light by a chemiluminescent analyzer.
At present, the chemiluminescent product kit is liquid-packed, and the core raw materials (antigen or antibody) used by the chemiluminescent product are bioactive substances, so that the bioactivity is easily lost at normal temperature, all the liquid kits are required to be transported by a cold chain and stored at low temperature, the operation is troublesome, in the prior art, the freeze-dried microspheres are adopted to replace liquid reagents, so that the problems of transportation and low-temperature storage in the prior art are solved, but the freeze-dried microspheres are generally harsh in storage conditions, the microspheres are easy to crack and have low hardness, the active ingredients of the reagents are unstable, the re-solubility is poor, and the like, so that the detection accuracy and the detection sensitivity are influenced.
Disclosure of Invention
The invention aims to provide a freeze-dried microsphere for luminescence immunoassay and a preparation method thereof, wherein the freeze-dried microsphere prepared by the preparation method is easy to store, has high hardness, is not easy to crack, has a good re-dissolution effect, and has stable active ingredients in a reagent in the use process, and the accuracy and the sensitivity of the detection are not affected.
In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:
the invention provides a preparation method of freeze-dried microspheres for luminescence immunoassay, which comprises the following steps:
mixing the prepared chemiluminescent immunoreagent with a protective agent in a volume ratio of 1:1.5, dripping liquid nitrogen into the mixture to form microspheres, and performing vacuum freeze drying on the microspheres to form freeze-dried microspheres;
wherein the protective agent comprises: trehalose, mannitol, soybean oil, 1,2 hexanediol and octanediol.
Preferably, as a further embodiment, 1 to 5 parts by mass of trehalose, 2 to 6 parts by mass of mannitol, 0.6 to 1.5 parts by mass of soybean oil, 0.8 to 1.2 parts by mass of 1,2 hexanediol and 0.1 to 0.5 parts by mass of octanediol are calculated.
Preferably, as a further embodiment, the trehalose is 2-4 parts by mass, mannitol is 3-5 parts by mass, soybean oil is 0.8-1.3 parts by mass, 1,2 hexanediol is 0.9-1.1 parts by mass, and octanediol is 0.2-0.4 parts by mass.
Preferably, as a further embodiment, 3 parts of trehalose, 4 parts of mannitol, 1.1 parts of soybean oil, 1.0 parts of 1,2 hexanediol and 0.3 parts of octanediol.
In the microsphere freeze-drying process, mannitol, trehalose, soybean oil, 1,2 hexanediol and octanediol are mixed with a chemiluminescent immunoreagent, so that the stability of active ingredients per se is improved, especially, 1,2 hexanediol and octanediol are added simultaneously, the stability effect of the active ingredients is synergistically enhanced, the influence of the surrounding environment on the microspheres per se is reduced, particularly, aggregation of some ingredients which are easy to combine with the active ingredients to cause deterioration on the surfaces of the microspheres is avoided, the microspheres are comprehensively protected, the addition of the ingredients does not influence the exertion of the detection effect of the reagents per se, especially, the small amount of soybean oil is compounded with the trehalose to play a synergistic effect, a non-hygroscopic protection layer is formed on the surfaces of the microspheres, the low-temperature protection and dehydration protection effects are realized, and the forming degree of the microspheres per se is improved.
Of course, through continuous practice, the specific components of the compound and the controlled dosage of each component are found to be optimized, especially the specific collocation of the 1,2 hexanediol and the octanediol on the basis of mannitol, and the specific combination of the soybean oil and the trehalose, if the soybean oil and the trehalose are changed into other components, the good synergistic effect cannot be generated. The adding aspect is also to make continuous fumbling according to the stability of the microsphere in the specific operation process, the adding amount of 1,2 hexanediol and octanediol relative to mannitol is much less, because the adding of the 1,2 hexanediol and the octanediol mainly has the protection effect on the active ingredients, if the adding amount is too large, the effect of the active ingredients is affected, and if the adding amount is too large, the adding amount of the soybean oil is as little as possible, only the moisture-resistant effect is achieved, and if the adding amount is too large, the degradation is difficult, and the normal use of the microsphere is also affected.
Preferably, as a further embodiment, sorbic acid and oxalic acid are added prior to lyophilization with liquid nitrogen.
Preferably, as a further embodiment, sorbic acid 0.2-0.9 parts by mass and oxalic acid 0.09-0.1 parts by mass.
In order to improve the re-dissolution effect of the freeze-dried microsphere, the protective agent is added, and the sorbic acid and the oxalic acid are added at the same time, so that the instant solubility of the microsphere can be accelerated by controlling the synergistic effect of the sorbic acid and the oxalic acid in a proper addition range, the microsphere can be rapidly dispersed into a uniform system, the dissolution efficiency is high, no solid slag exists after the dissolution, the dispersion is uniform, the use is very convenient, the working efficiency is improved, and the detection efficiency is also improved.
Preferably, as a further embodiment, the procedure of freeze-drying is carried out in multiple stages, wherein the time of the first freeze-drying stage is 1-10h and the temperature is-40 to-55 ℃.
Preferably, as a further embodiment, the procedure of freeze-drying is carried out in three stages, the second freeze-drying stage being carried out at a temperature of from-30 to-35℃and the third freeze-drying stage being carried out at a temperature of from 20 to 40℃for a time of from 4 to 7 hours
Preferably, as a further embodiment, the procedure of freeze-drying is carried out under vacuum of 70-120 ubar.
In order to improve the stability of the microsphere, the invention carries out the procedural control on the whole freeze-drying process, and is divided into a plurality of stages, and the temperature and the freeze-drying time of each stage are adjusted, so that the active ingredients of the whole microsphere are not easy to run off, are not easy to crack and have good formability in the freeze-drying process by accurately controlling the temperature and the time.
In a word, the freeze-dried microsphere prepared by the preparation method has better forming degree and better subsequent use effect.
Compared with the prior art, the invention has the beneficial effects that:
(1) The reagent and the operation parameters related to the microsphere freeze-drying process are controlled more accurately, so that the obtained freeze-dried microsphere is easy to store, and the microsphere is high in hardness and not easy to crack.
(2) The microsphere prepared by the preparation method of the freeze-dried microsphere can be rapidly dispersed into a uniform system, is convenient to use, improves the working efficiency and also improves the detection efficiency.
(3) The microsphere prepared by the microsphere freeze-drying process disclosed by the invention is not easy to influence the quality of the microsphere due to oxygen even if the microsphere is placed and stored in an air atmosphere, and the microsphere is good in stability and good in moisture resistance.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1
The microsphere freeze-drying preparation method for the luminescence immunoassay comprises the following steps:
(1) The protective agent is prepared in advance: stirring and dissolving 1g of trehalose, 6g of mannitol, 0.6g of soybean oil, 1.2g of 1,2 hexanediol, 0.1g of octanol, 0.2g of sorbic acid, 0.1g of oxalic acid and 1000ml of purified water, and filtering the formed solution by adopting a filter membrane;
(2) Adding the prepared luminous immunoreagent into the protective agent in the step (1), wherein the volume ratio of the luminous immunoreagent to the active ingredients of the protective agent is 1:1.5;
(3) Dripping liquid nitrogen into the mixture to form microspheres, and performing vacuum freeze-drying on the microspheres to form freeze-dried microspheres;
(4) The procedure of freeze drying is carried out in three stages, wherein the time of the first freeze drying stage is 1h, the temperature is-55 ℃, the time of the second freeze drying stage is 8h, the temperature is-30 ℃, the time of the third freeze drying stage is 7h, the temperature is 20 ℃, and the vacuum degree is 70-120 ubar;
(5) The freeze-dried microsphere obtained by the steps is stored in a sealing way, and can be directly applied to chemiluminescence immunodetection later.
Example 2
The microsphere freeze-drying preparation method for the luminescence immunoassay comprises the following steps:
(1) The protective agent is prepared in advance: 5g of trehalose, 2g of mannitol, 1.5g of soybean oil, 0.8g of 1,2 hexanediol, 0.5g of octanol, 0.9g of sorbic acid, 0.09g of oxalic acid and 1000ml of purified water are stirred and dissolved, and a filter membrane is adopted to filter the formed solution;
(2) Adding the prepared luminous immunoreagent into the protective agent in the step (1), wherein the volume ratio of the luminous immunoreagent to the active ingredients of the protective agent is 1:1.5;
(3) Dripping liquid nitrogen into the mixture to form microspheres, and performing vacuum freeze-drying on the microspheres to form freeze-dried microspheres;
(4) The procedure of freeze drying is carried out in three stages, wherein the time of the first freeze drying stage is 10 hours, the temperature is-40 ℃, the time of the second freeze drying stage is 2 hours, the temperature is-35 ℃, the time of the third freeze drying stage is 4 hours, the temperature is 40 ℃, and the vacuum degree is 70-120 ubar;
(5) The freeze-dried microsphere obtained by the steps is stored in a sealing way, and can be directly applied to chemiluminescence immunodetection later.
Example 3
The microsphere freeze-drying preparation method for the luminescence immunoassay comprises the following steps:
(1) The protective agent is prepared in advance: 2g of trehalose, 5g of mannitol, 0.8g of soybean oil, 1.1g of 1,2 hexanediol, 0.2g of octanol, 0.9g of sorbic acid, 0.09g of oxalic acid and 1000ml of purified water are stirred and dissolved, and a filter membrane is adopted to filter the formed solution;
(2) Adding the prepared luminous immunoreagent into the protective agent in the step (1), wherein the volume ratio of the luminous immunoreagent to the active ingredients of the protective agent is 1:1.5;
(3) Dripping liquid nitrogen into the mixture to form microspheres, and performing vacuum freeze-drying on the microspheres to form freeze-dried microspheres;
(4) The procedure of freeze drying is carried out in three stages, wherein the time of the first freeze drying stage is 7 hours, the temperature is-45 ℃, the time of the second freeze drying stage is 6 hours, the temperature is-32 ℃, the time of the third freeze drying stage is 5 hours, the temperature is 30 ℃, and the vacuum degree is 70-120 ubar;
(5) The freeze-dried microsphere obtained by the steps is stored in a sealing way, and can be directly applied to chemiluminescence immunodetection later.
Example 4
The specific procedure was as in example 3, except that: trehalose 4g, mannitol 3g, soybean oil 1.3g, 1,2 hexanediol 0.9g, octanol 0.4g, sorbic acid 0.7g, oxalic acid 0.095g and purified water 1000ml were dissolved by stirring.
Example 5
The specific procedure was as in example 3, except that: trehalose 3g, mannitol 4g, soybean oil 1.1g, 1,2 hexanediol 1.0g, octanol 0.3g, sorbic acid 0.7g, oxalic acid 0.095g and purified water 1000ml were dissolved by stirring.
Example 6
The specific procedure was as in example 3, except that no sorbic acid and no oxalic acid were added.
Example 7
The specific procedure is as in example 3, except that 1g of octanol is used.
Example 8
The specific procedure is as in example 3, except that 1.5g of hexanediol is used.
Comparative example 1
The specific procedure is as in example 3, except that hexanediol is converted to ethylene glycol.
Comparative example 2
The specific procedure was as in example 3, except that no soybean oil was added.
Example 9
The specific procedure was as in example 3, except that the procedure of freeze-drying was carried out in two stages, the first freeze-drying stage being carried out at a temperature of-45℃and the second freeze-drying stage being carried out at a temperature of 30℃and a vacuum of between 50 and 150 ubar.
Experimental example 1
The stability of the microspheres of each of the above examples and comparative examples was confirmed by the change in the moisture absorption rate of the microspheres when left for 24 hours at different ambient humidity in an air environment and the appearance thereof when exposed to air environment conditions at different temperatures for 48 hours, wherein the dry environment means that the ambient humidity is 3% or less:
table 1 experimental results
As can be seen from the data in table 1 above, the microspheres prepared in each example and comparative example were exposed to a humid air environment, and although the moisture absorption rate thereof tended to increase according to the increase in ambient humidity, the stability of the microspheres themselves was also different since the operating conditions of each example and comparative example were different throughout the implementation of the lyophilization process, and from the data on the moisture absorption rate, the optimum process was example 5, and example 5 was controlled to be optimum in terms of both the added dimensionally stable components and the lyophilization operating conditions, and therefore the increase trend of the moisture absorption rate and moisture absorption rate was low, indicating that the microspheres of this example were still able to achieve good stability and moisture resistance even when exposed to a humid air environment, were relatively slightly different from those of examples 5, but were relatively poor in stability, and from example 6 onwards, since sorbic acid and oxalic acid were added to mainly increase the re-solubility, the two substances were not added, and were not well formed, and were relatively poor in uniformity of dissolution rate and poor in dissolution after formation of a relatively rough surface. As can be seen from the data of examples 7 and 8, the addition of alcohol also has a certain dosage matching relationship, if the addition of octaethylene glycol and hexanediol is too large, the compatibility relationship between the octaethylene glycol and mannitol is affected, the stability of the active ingredient is poor, the moisture absorption rate is easy to be affected by the environment and is fast to rise, in addition, as can be seen from the data of comparative example 2, the moisture resistance of the microsphere is affected after no soybean oil is added, and the stability effect of the final microsphere is affected, and although the addition of soybean oil is relatively large, the soybean oil can form a good compounding relationship with other substances after the addition of the soybean oil, so that the moisture resistance is ensured, but the moisture absorption rate is fast to rise when the soybean oil is not added. In comparative example 1, since the type of alcohol is changed to other types, the effect is not as expected, and therefore, the fixed collocation and the specific proportioning amount are all important guarantees of the stability of the microsphere in the scheme of the invention. Final example 9 also has an effect on the final moisture absorption rate since the lyophilization process is not controlled to be optimal.
As can be seen from the changes in the appearance of the microspheres at different temperatures in the table, the preferred embodiment is example 5, and the other embodiments are affected to some extent from example 6 because the stability of the microspheres is poor.
In summary, as can be seen from the data in table 1 above, the lyophilization process of the present invention requires that each operating condition be controlled within the optimal range to achieve good results.
While particular embodiments of the present invention have been illustrated and described, it will be appreciated that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
Claims (10)
1. The preparation method of the freeze-dried microsphere for luminescence immunoassay is characterized by comprising the following steps of:
mixing the prepared chemiluminescent immunoreagent with a protective agent in a volume ratio of 1:1.5, dripping liquid nitrogen into the mixture to form microspheres, and performing vacuum freeze drying on the microspheres to form freeze-dried microspheres;
wherein the protective agent comprises: trehalose, mannitol, soybean oil, 1,2 hexanediol and octanediol.
2. The preparation method according to claim 1, wherein the trehalose is 1-5 parts by mass, the mannitol is 2-6 parts by mass, the soybean oil is 0.6-1.5 parts by mass, the 1, 2-hexanediol is 0.8-1.2 parts by mass, and the octanediol is 0.1-0.5 parts by mass.
3. The preparation method according to claim 1, wherein the trehalose is 2-4 parts by mass, the mannitol is 3-5 parts by mass, the soybean oil is 0.8-1.3 parts by mass, the 1, 2-hexanediol is 0.9-1.1 parts by mass, and the octanediol is 0.2-0.4 parts by mass.
4. The preparation method according to claim 1, wherein the trehalose comprises 3 parts by mass, 4 parts by mass, 1.1 parts by mass of soybean oil, 1.0 parts by mass of 1, 2-hexanediol and 0.3 parts by mass of octanediol.
5. The method of claim 1, wherein sorbic acid and oxalic acid are added prior to lyophilization with liquid nitrogen.
6. The preparation method according to claim 5, wherein the sorbic acid is 0.2-0.9 parts and the oxalic acid is 0.09-0.1 parts by mass.
7. The process according to any one of claims 1 to 6, wherein the lyophilization process is carried out in multiple stages, wherein the first lyophilization stage is carried out for a period of 1 to 10 hours and at a temperature of-40 to-55 ℃.
8. The process according to claim 7, wherein the freeze-drying procedure is carried out in three stages, the second freeze-drying stage being carried out at a temperature of-30 to-35 ℃ for 2-8 hours and the third freeze-drying stage being carried out at a temperature of 20-40 ℃ for 4-7 hours.
9. The method according to claim 7, wherein the procedure of freeze-drying is performed under a vacuum of 70 to 120 ubar.
10. A lyophilized microsphere obtained by the method of any one of claims 1-9.
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