Protein-microsphere conjugate storage solution and application thereof

Abstract

The invention provides a protein-microsphere conjugate storage solution and application thereof. The protein-microsphere conjugate storage solution comprises 95-105 mM of citric acid-sodium citrate buffer solution; and on the basis of the mass of the storage liquid, the storage liquid further comprises the following components in percentage by mass: 0.5-1.5% of inorganic salt; 0.08%-0.12% of a preservative; 0.5%-1.5% of protective protein; the pH (Potential of Hydrogen) value of the storage solution is 6.0 to 6.5.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological immunology, and in particular relates to a protein-microsphere conjugate storage solution and application thereof. Background Art

[0002] Light Initiated Chemiluminescent Assay is a homogeneous immunoassay technology that relies on the close proximity of two particles to complete chemical energy transfer. This technology mainly uses two receptor / donor nanospheres coated with antibodies or antigens, which specifically bind under suitable conditions. The donor microspheres are coated with photosensitive substances such as phthalocyanine. Under 680nm laser irradiation, singlet oxygen is generated. The singlet oxygen transmits the signal to the receptor microspheres within the range of 200nm, and reacts chemically with the dimethylthiophene derivatives therein to generate ultraviolet light that is finally converted into light signals. In this technology, each component is uniform in the system and the entire reaction process does not require cleaning and separation processes, which reduces the error of the reaction and can complete the chemiluminescence reaction more quickly and fully.

[0003] At present, the technology model is relatively mature, but in the application process, it is inevitable that some microsphere-protein conjugates need to be stored for a long time. The conjugate is stored at a high concentration at 2-8°C, which is prone to aggregation and sedimentation. It is found that in the subsequent chemiluminescence detection, this phenomenon will cause obvious changes in light values, resulting in large detection errors, and reducing the stability and reliability of detection data.

[0004] The current storage solution formula mainly includes a buffer system and a preservative, and the protection of the protein-microsphere conjugate is still poor. Summary of the invention

[0005] The present disclosure provides a protein-microsphere conjugate storage solution and application thereof, which can effectively solve the above problems.

[0006] The present disclosure is implemented as follows:

[0007] In a first aspect, the present disclosure provides a protein-microsphere conjugate storage solution, the storage solution comprising: 95-105 mM citric acid-sodium citrate buffer;

[0008] And, based on the mass of the storage solution, the storage solution also includes the following components in mass fractions: 0.5% to 1.5% inorganic salt; 0.08% to 0.12% preservative; 0.5% to 1.5% protective protein;

[0009] The pH value of the storage solution is 6.0-6.5.

[0010] In a second aspect, the present disclosure provides an application of the storage solution described in the first aspect in a dispersed protein-microsphere conjugate, characterized in that the protein-microsphere conjugate is repeatedly cleaned with a cleaning solution containing a surfactant and then added to the storage solution for storage at a storage temperature of 2°C to 8°C.

[0011] In a third aspect, the present disclosure provides a method for storing a protein-microsphere conjugate, wherein the protein-microsphere conjugate is repeatedly cleaned with a cleaning solution containing a surfactant, and then added to the storage solution described in the first aspect for storage at a storage temperature of 2°C to 8°C.

[0012] The beneficial effects of the present disclosure are:

[0013] The present disclosure provides a storage solution for a protein-microsphere conjugate, which can slow down the aggregation between the conjugates and keep the conjugates in a uniformly suspended state in the solution, thereby improving the signal stability of the photoluminescence of the conjugates and making the results of subsequent chemiluminescence detection more stable and reliable.

[0014] Furthermore, the protein-microsphere conjugate was stored in the storage solution at 2-8° C. for 35 days without ultrasonication, and the luminescence value of chemiluminescence detection after sufficient mixing remained stable. When stored for 30 days, the luminescence value of chemiluminescence detection after sufficient mixing was slightly different from the luminescence value of chemiluminescence detection after complete ultrasonication. DETAILED DESCRIPTION

[0015] In order to make the purpose, technical solutions and advantages of the embodiments of the present disclosure clearer, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present disclosure, rather than all of the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present disclosure. Therefore, the following detailed description of the embodiments of the present disclosure is not intended to limit the scope of the present disclosure for protection, but merely represents the selected embodiments of the present disclosure. Based on the embodiments in the present disclosure, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present disclosure.

[0016] The disclosed embodiment provides a protein-microsphere conjugate storage solution, the storage solution comprising: 95-105 mM citric acid-sodium citrate buffer;

[0017] And, based on the mass of the storage solution, the storage solution also includes the following components in mass fractions: 0.5% to 1.5% inorganic salt; 0.08% to 0.12% preservative; 0.5% to 1.5% protective protein;

[0018] The pH value of the storage solution is 6.0-6.5.

[0019] The aggregation phenomenon of protein-coated microspheres is speculated to be caused by nonspecific adsorption, protein inactivation, etc.

[0020] From the perspective of the storage solution of protein-microsphere conjugates, affected by the external medium, as the storage time goes by, the surface charge of the coupled protein in the conjugate will change, resulting in the repulsion between the microspheres may be insufficient to resist the gravitational force; in addition, other proteins in the solution will also be more easily adsorbed to the surface of the microspheres, resulting in non-specific adsorption. As more proteins are adsorbed, the molecules become closer, causing the conformation of the protein to change and become inactive.

[0021] The storage solution provided in the embodiment of the present disclosure uses a citric acid-sodium citrate buffer system. Compared with other buffer systems, such as TRIS, HEPES, MES, sodium dihydrogen phosphate-disodium hydrogen phosphate, the storage solution components provided in the embodiment of the present disclosure are conducive to stabilizing the surface structure of the microspheres coated with proteins, and can better alleviate the aggregation between the microspheres, and avoid sedimentation of the microspheres after aggregation. Moreover, the charge conditions in the weakly acidic solution system have a good protective effect on the protein.

[0022] In protein structure studies, sodium citrate-citric acid buffer system can provide a suitable environment to help maintain the native conformation and activity of proteins.

[0023] In some embodiments, the inorganic salt is at least one of sodium chloride, magnesium chloride, and potassium chloride.

[0024] The inorganic salt in the solution system can effectively adjust the ion concentration of the system, and in a single factor experiment, it can be observed with the naked eye that as the concentration of the inorganic salt increases, the sedimentation of the microspheres slows down.

[0025] However, too low an inorganic salt concentration cannot balance the repulsive force between the surface charges of the microspheres, while too high an inorganic salt ion concentration will weaken the double electrical layer structure outside the microspheres and accelerate the aggregation of the microspheres.

[0026] In some embodiments, the preservative is at least one of PC300 or PC950.

[0027] In some embodiments, based on the mass of the storage solution, the storage solution further comprises the following components in mass fractions: 1.2% to 1.5% of protective protein.

[0028] Within a limited concentration range, a storage solution containing a high concentration of protective protein will have a better effect in alleviating the sedimentation of the protein-microsphere conjugate.

[0029] In some embodiments, the protective protein is bovine serum albumin.

[0030] In some embodiments, the bovine serum albumin is precision grade bovine serum albumin.

[0031] The precision-grade bovine serum albumin contains high-content monomers and ultra-low-content fatty acids, does not contain immunoglobulin G (IgG) and protease, and the purity of the bovine serum albumin is greater than or equal to 98%.

[0032] Wherein, the mass content of non-esterified fatty acid NEFA in the protective protein is not more than 0.01%.

[0033] Non-esterified fatty acids are composed of long-chain hydrocarbons and carboxylic acid groups (-COOH), of which the hydrocarbon chain is non-polar and therefore hydrophobic. Due to its high hydrophobicity, NEFA may interact with proteins, affecting their structure and function, thereby exacerbating the sedimentation tendency of protein-coated microspheres.

[0034] The protective protein in the solution system can maintain sufficient and stable closure of the surface active sites of the coupled protein in the protein-microsphere conjugate. Appropriate concentration considerations can reduce the active sites from being snatched by other proteins and reduce nonspecific adsorption, thereby ensuring a stable state between the microsphere conjugates for a long time, uniform suspension, reducing changes in the properties of the microsphere conjugates caused by aggregation, and maintaining stability in light value detection.

[0035] Moreover, under weakly acidic conditions, the stability of the blocking effect of the protective protein on the surface active sites of the coupled protein in the protein-microsphere conjugate can be improved.

[0036] In some embodiments, based on the mass of the storage solution, the storage solution further comprises the following components in mass fractions: 4% to 6% of sugars.

[0037] In some embodiments, the sugar is at least one of sucrose, trehalose, glucose, and dextran.

[0038] Adding an appropriate amount of sugar to the solution system can adjust the system density and maintain the uniform suspension state of the microspheres in the solution.

[0039] Furthermore, a certain concentration of sugars can also protect the surface active sites of the coupled protein in the protein-microsphere conjugate, reduce nonspecific adsorption, and the phenomenon of protein inactivation caused by the increase of storage time, and can better slow down the aggregation between the microspheres and maintain the uniform suspension state of the microspheres, that is, the sugars and protective proteins in the solution system have a synergistic effect.

[0040] The storage solution provided in the embodiments of the present disclosure can better slow down the aggregation of microspheres and maintain the uniform suspension of microspheres by adjusting the ion concentration of the system through inorganic salt ions under appropriate pH conditions to balance the repulsive force of the surface charges between microspheres.

[0041] Moreover, through the synergistic effect of each component, the signal stability of the protein-microsphere conjugate can be significantly improved, making the results of subsequent chemiluminescence detection more stable and reliable, thereby solving the storage problem of the protein-microsphere conjugate.

[0042] In some embodiments, the protein in the protein-microsphere conjugate is an antibody protein or an antigen protein.

[0043] In some embodiments, the protein in the protein-microsphere conjugate includes at least one of an antibody against creatine kinase isoenzyme CK-MB, an antibody against myoglobin MYO, and an antibody against amino-terminal pro-brain natriuretic peptide NT-proBNP.

[0044] The storage solution provided in the embodiments of the present disclosure has the same performance of slowing down the aggregation phenomenon between microspheres on different protein-microsphere conjugates.

[0045] The disclosed embodiment provides an application of the above storage solution in dispersed protein-microsphere conjugates. The protein-microsphere conjugates are repeatedly cleaned with a cleaning solution containing a surfactant and then added to the storage solution for storage at a storage temperature of 2°C to 8°C.

[0046] In some embodiments, the protein-microsphere conjugate after coupling is centrifuged, the supernatant is discarded, and then ultrasonically resuspended with a 0.2% by mass TW-20 solution, and the washing is repeated twice before adding to the storage solution for storage.

[0047] In some embodiments, the application is the application of the storage solution in a chemiluminescent detection reagent, and the chemiluminescent detection reagent includes the protein-microsphere conjugate stored in the storage solution.

[0048] In some embodiments, the reagents are mixed thoroughly before being used for chemiluminescent detection.

[0049] The disclosed embodiment provides a method for storing a protein-microsphere conjugate. The protein-microsphere conjugate is repeatedly cleaned with a cleaning solution containing a surfactant, and then added to the above storage solution for storage at a storage temperature of 2°C to 8°C.

[0050] In some embodiments, the cleaning solution containing a surfactant is a TW-20 solution with a mass fraction of 0.2%.

[0051] Example 1

[0052] Sources of reagents in the disclosed embodiments:

[0053] Bovine serum albumin 1 was purchased from Proliant, catalog number: 69760.

[0054] Bovine serum albumin 2 was purchased from Sigma, catalog number: B2064.

[0055] Casein was purchased from Sigma.

[0056] The nanoparticles are carboxyl microspheres purchased from.

[0057] Creatine kinase isoenzyme antibodies were purchased from Boyue Biological Co., Ltd.

[0058] Myoglobin antibody was purchased from Wuhan Aokebote Co., Ltd.

[0059] The amino-terminal pro-brain natriuretic peptide antibody was purchased from Feipeng Biotechnology Co., Ltd.

[0060] The first group of storage solution configuration:

[0061] The concentration of citric acid-sodium citrate is 100 mM. Based on the mass of the storage solution, the storage solution further comprises the following components in mass fractions: sodium chloride 0.9%; preservative PC300 0.1%.

[0062] The above components are dissolved in pure water, and the pH of the solution is initially adjusted to about 6.5. Then, bovine serum albumin 1 is added to the solution and dissolved evenly. The pH of the solution is fine-tuned to 6.5 again, and pure water is added to make up the volume. After stirring evenly, the storage solution is obtained by filtering.

[0063] Based on the mass of the storage solution, the added amount of bovine serum albumin 1 is 1.5%.

[0064] The second group of storage solution configuration:

[0065] The difference from the first group of stock solutions is that sucrose is added and dissolved in pure water in the same step as citric acid-sodium citrate.

[0066] Based on the mass of the storage solution, the added amount of sucrose is 5%.

[0067] The third group of storage solution configuration:

[0068] The difference from the first group of storage solutions is that before adding bovine serum albumin 1, the pH of the solution is preliminarily adjusted to about 6.0, and after adding bovine serum albumin 1, the pH of the solution is fine-tuned to 6.0 again.

[0069] The fourth group of storage solution configuration:

[0070] The difference from the first group of storage solutions is that the added amount of bovine serum albumin 1 is 0.5% based on the mass of the storage solution.

[0071] The fifth group of storage solution configuration:

[0072] The difference from the first group of storage solutions is that bovine serum albumin 1 in the storage solution is replaced by bovine serum albumin 2.

[0073] Based on the mass of the storage solution, the added amount of bovine serum albumin 2 is 1.5%.

[0074] The sixth group of storage solution configuration:

[0075] The difference from the first group of storage solutions is that bovine serum albumin 1 in the storage solution is replaced by casein.

[0076] Based on the mass of the storage solution, the added amount of casein is 0.5%.

[0077] The seventh group of storage solution configuration:

[0078] The difference from the first group of stock solutions is that the buffer solution in the stock solutions is replaced with 50 mM Tris solution.

[0079] And, the bovine serum albumin 1 in the stock solution was replaced with bovine serum albumin 2. Before adding the bovine serum albumin 2, the pH of the solution was initially adjusted to about 7.4, and after adding the bovine serum albumin 2, the pH of the solution was fine-tuned to 7.4 again.

[0080] Based on the mass of the storage solution, the added amount of bovine serum albumin 2 is 1.5%.

[0081] The eighth group of storage solution configuration:

[0082] The difference from the first group of storage solutions is that the buffer solution in the storage solution is replaced with 200 mM disodium hydrogen phosphate-sodium dihydrogen phosphate solution, and before adding bovine serum albumin 1, the pH of the solution is initially adjusted to about 7.4, and after adding bovine serum albumin 1, the pH of the solution is fine-tuned to 7.4 again.

[0083] Example 2

[0084] The protein-microsphere conjugate after coupling was centrifuged, the supernatant was discarded, and it was resuspended by ultrasound with a 0.2% TW-20 solution. After repeated washing twice, the eight groups of storage solutions in Example 1 were added respectively for storage at a storage temperature of 4°C.

[0085] In each set of storage solution, the concentration of the protein-microsphere conjugate was configured to be 5 mg / mL.

[0086] The suspension state of the protein-microsphere conjugate in the storage solution was observed with the naked eye, and the state of each group of storage solution after standing for 7 days, 14 days, and 30 days was recorded.

[0087] The experimental results of the protein-microsphere conjugates were CK-MB antibody-microsphere conjugate, MYO antibody-microsphere conjugate and NT-proBNP antibody-microsphere conjugate, respectively. The results are shown in Tables 1 to 3.

[0088] Table 1 Suspension state of CK-MB antibody-microsphere conjugate

[0089]

[0090] Table 2 MYO antibody-microsphere conjugate suspension state

[0091]

[0092] Table 3 Floating state of NTroBNP antibody-microsphere conjugate

[0093]

[0094] As can be seen from Tables 1 and 2, after storing the three types of microspheres in the first, second, third and sixth groups of storage solutions for 35 days, no sedimentation occurred and the microspheres were evenly dispersed in the solution, indicating that these four groups of storage solutions have a better effect in alleviating sedimentation.

[0095] In particular, for NT-proBNP antibody-microsphere conjugates that are not suitable for storage under acidic conditions in traditional storage methods, these four groups of storage solutions can also make them evenly dispersed in the solution for a long time.

[0096] Compared with the fifth group of storage solution, the NT-proBNP antibody-microsphere conjugate stored in the storage solution showed obvious sedimentation after 18 days. Conventional bovine serum albumin 2 had no way to alleviate its sedimentation rate, but the selection of relatively high-purity bovine serum albumin 1 could effectively protect the surface electrical layer structure of the protein-microsphere conjugate, thereby better preventing protein aggregation.

[0097] The fourth group of storage solutions stored MYO antibody-microsphere conjugates and NT-proBNP antibody-microsphere conjugates respectively, and slight sedimentation occurred after 18 days, indicating that the storage solution provided by the present disclosure preferably contains a protective protein with a mass fraction of 1.5%.

[0098] The storage solution containing high concentration of protective protein will have a better effect in alleviating the sedimentation of protein-microsphere conjugates.

[0099] The eighth group of storage solutions were used to store CK-MB antibody-microsphere conjugates and MYO antibody-microsphere conjugates, and slight sedimentation occurred after 18 days, indicating that the storage solution used a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer system, which was not as effective as the citric acid-sodium citrate buffer system for storing protein-microsphere conjugates.

[0100] Example 3

[0101] The protein-microsphere conjugate after coupling was centrifuged, the supernatant was discarded, and it was resuspended by ultrasound with a 0.2% TW-20 solution. After repeated washing twice, the eight groups of storage solutions in Example 1 were added respectively for storage at a storage temperature of 4°C.

[0102] The concentration of protein-microsphere conjugates was configured to be 5 mg / mL.

[0103] Each set of solutions containing protein-microsphere conjugates was equally divided into four parts, which were recorded as samples one to four.

[0104] Samples 1 to 4 were left to stand for 0 d, 7 d, 18 d and 30 d respectively.

[0105] After each sample has been allowed to stand for a predetermined time, it is completely sonicated and mixed, and chemiluminescence detection is performed under the same conditions to record the chemiluminescence values ​​of the gradient sample concentrations.

[0106] Wherein, the sample is a plasma sample containing the corresponding antigen.

[0107] The experimental results of the protein-microsphere conjugates were CK-MB antibody-microsphere conjugate, MYO antibody-microsphere conjugate and NT-proBNP antibody-microsphere conjugate, respectively. The results are shown in Tables 4 to 6.

[0108] Table 4 Luminescence value of CK-MB antibody-microsphere conjugate

[0109]

[0110]

[0111] Table 5 Luminescence values ​​of MYO antibody microsphere conjugates

[0112]

[0113]

[0114] Table 6 Luminescence value of NT-proBNP antibody-microsphere conjugate

[0115]

[0116]

[0117] It can be seen from Tables 4 to 6 that, taking the detection luminescence value of the seventh group of storage solutions as a benchmark, the detection luminescence values ​​of the samples of each concentration in the first, second, third and fourth groups are more stable as the storage time increases, indicating that the effect of these four groups of storage solutions in improving the signal stability of the protein-microsphere conjugates is better than the conventional TRIS buffer system method, and the preferred storage solution components of the embodiments of the present disclosure can better maintain the detection activity of the microspheres.

[0118] In the fifth and sixth groups, as the storage time increases, the stability of the signal value is relatively poor compared with the conventional storage solution in the seventh group. Excluding the influence of environmental factors, it is further verified that the bovine serum albumin 1 in the preferred storage solution component of the embodiment of the present disclosure can significantly protect the surface active sites of the protein-microsphere conjugate and reduce its nonspecific adsorption.

[0119] Compared with the conventional disodium hydrogen phosphate-sodium dihydrogen phosphate buffer system, the citric acid-sodium citrate buffer system used in the embodiment of the present disclosure is better in terms of storage effect.

[0120] Example 4

[0121] The protein-microsphere conjugates after coupling were centrifuged, the supernatant was discarded, and then resuspended with 0.2% TW-20 solution, washed twice, and then added to the eight groups of storage solutions in Example 1 for storage at a storage temperature of 4°C.

[0122] The concentration of protein-microsphere conjugates was configured to be 5 mg / mL.

[0123] Each stock solution was divided into two parts, which were named samples five and six.

[0124] Samples 5 and 6 were left to stand for 15 d and 30 d respectively.

[0125] After each sample has been left to stand for a predetermined time, it is first subjected to only a pre-treatment by mixing, and then subjected to chemiluminescence detection under the same conditions.

[0126] Then, the sample was completely sonicated and mixed, and chemiluminescence detection was performed under the same conditions to record the chemiluminescence values ​​of the gradient sample concentrations.

[0127] The two sets of test data were collated for comparison.

[0128] The experimental results of the protein-microsphere conjugates were CK-MB antibody-microsphere conjugate, MYO antibody-microsphere conjugate and NT-proBNP antibody-microsphere conjugate, respectively. The results are shown in Tables 7 to 9.

[0129] Table 7 Comparison of luminescence values ​​of CK-MB antibody microsphere conjugates

[0130]

[0131] Table 8 Comparison of luminescence values ​​of MYO antibody-microsphere conjugates

[0132]

[0133] Table 9 Comparison of luminescence values ​​of NT-proBNP antibody-microsphere conjugates

[0134]

[0135] It can be seen from Tables 7 to 9 that after storage for 30 days using the storage solution involved in the present disclosure, the difference in luminescence values ​​detected after only mixing and complete sonication is small; this indicates that the protein and microsphere conjugates stored in the preferred storage solution formula of the embodiments of the present disclosure can achieve a state in which the conjugates are uniformly dispersed in the solution within a certain period of time, reducing aggregation caused by uneven charge distribution during long-term storage, and can be used as a preferred storage solution for protein-microsphere conjugates in related platforms.

[0136] The above description is only a preferred embodiment of the present disclosure and is not intended to limit the present disclosure. For those skilled in the art, the present disclosure may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present disclosure shall be included in the protection scope of the present disclosure.

Claims

1. A protein-microsphere conjugate storage solution, characterized in that: The storage solution includes: 95-105 mM citric acid-sodium citrate buffer; And, based on the mass of the storage solution, the storage solution also includes the following components in mass fractions: 0.5% to 1.5% inorganic salt; 0.08% to 0.12% preservative; 0.5% to 1.5% protective protein; The pH value of the storage solution is 6.0-6.

5.

2. The storage solution according to claim 1, characterized in that Based on the mass of the storage solution, the storage solution also includes the following components in mass fractions: 4% to 6% of sugars.

3. The storage solution according to claim 1, characterized in that The protein in the protein-microsphere conjugate includes at least one of creatine kinase isoenzyme antibody, myoglobin antibody, and amino-terminal pro-brain natriuretic peptide antibody.

4. The storage solution according to claim 1, characterized in that The protective protein is bovine serum albumin.

5. The storage solution according to claim 1, characterized in that The mass content of non-esterified fatty acids in the protective protein is no more than 0.01%.

6. Use of the storage solution according to any one of claims 1 to 5 in a dispersed protein-microsphere conjugate, characterized in that: The protein-microsphere conjugate is repeatedly washed with a washing solution containing a surfactant, and then added to the storage solution for storage at a storage temperature of 2°C to 8°C.

7. The use according to claim 6, characterized in that The application is the application of the storage solution in a chemiluminescent detection reagent, and the chemiluminescent detection reagent includes the protein-microsphere conjugate stored in the storage solution.

8. The use according to claim 7, characterized in that After the reagents are fully mixed, they are used for chemiluminescence detection.

9. A method for storing a protein-microsphere conjugate, characterized in that: The protein-microsphere conjugate is repeatedly washed with a washing solution containing a surfactant, and then added to the storage solution according to any one of claims 1 to 5 for storage at a storage temperature of 2°C to 8°C.

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