Method for controlling batch-to-batch difference and improving specificity by changing raw material adding time after microsphere activation

Abstract

The invention provides a method for controlling batch-to-batch difference and improving specificity by changing raw material adding time after microsphere activation, and belongs to the technical field of biomedicine. The bioactive substances are added for coupling after the microspheres are activated for a certain time. According to the method, extra reagents or steps do not need to be introduced, batch-to-batch difference reduction and false positive reduction can be achieved only by optimizing the coupling object adding time, operation is easy, control is easy, reduction of false positive results caused by non-specific binding is facilitated, biological coupling reaction is more sensitive and reliable, and the method is suitable for industrial production. Therefore, the detection sensitivity, specificity and accuracy can be improved, and the kit has extremely high practical value in the fields which need large-scale production and have high requirements on product quality. The method provided by the invention can provide a more reliable and stable tool for biomedical research and clinical diagnosis, and has a good application prospect.

Description

Technical Field

[0001] The invention relates to the field of biomedical technology, and in particular to a method for controlling batch differences and improving specificity by changing the time of adding raw materials after microsphere activation. Background Art

[0002] In biomedical research and clinical diagnosis, carboxyl-modified microspheres are important carrier tools in medical and molecular biology research. The carboxyl functional groups on their surface can covalently couple biological ligands such as peptides, proteins, antibodies, and oligonucleotides to the surface of the microspheres under the action of special chemical reagents, thereby quickly classifying and enriching the target from the sample. It can be applied to chemiluminescence technology, flow fluorescence technology, chromatography technology and other fields.

[0003] At present, the activation of carboxyl-modified microspheres mainly relies on coupling agents such as EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide), which can effectively convert the carboxyl groups on the surface of the microspheres into highly reactive intermediates, thereby coupling with amino groups and other groups on biological molecules. However, in the existing carboxyl-modified microsphere labeling process, biological molecules are usually added immediately after the activation reaction for coupling. However, since the activation reaction is affected by many factors, the reactivity of microspheres after activation in different batches may vary greatly, resulting in inconsistent coupling efficiency of biological molecules in the subsequent labeling process, and then batch differences; and if the activated intermediates are unstable, they are easy to react with nonspecific biological molecules in the system to form nonspecific binding, resulting in the occurrence of false positives.

[0004] In order to improve the accuracy and repeatability of immunoassays, technicians in this field have been exploring new methods to control the activation and coupling process of magnetic microspheres, such as optimizing the buffer system, using more purified raw materials, and improving coupling technology, but these methods often cannot fundamentally solve the problems of batch differences and false positives. It can be seen that the existing magnetic microsphere activation and coupling technology still faces challenges in terms of batch consistency and specific binding. Therefore, it is necessary to provide a method that can solve the problems of large batch differences and high false positives in the labeling process of carboxyl-modified microspheres, improve the specificity and accuracy of labeling, reduce the occurrence of nonspecific binding, and provide a more reliable and stable tool for biomedical research and clinical diagnosis. Summary of the invention

[0005] The purpose of the present invention is to provide a method for controlling batch differences and improving specificity by changing the time of adding raw materials after microsphere activation, so as to solve the technical problems of large batch differences and high false positives in the microsphere activation and coupling process.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a method for controlling batch differences and improving specificity by changing the time of adding raw materials after microsphere activation, wherein biologically active substances are added for coupling after a certain period of time after microsphere activation.

[0007] Preferably, the microspheres include carboxyl-modified microspheres.

[0008] Preferably, the bioactive substance is added for coupling 18 to 30 minutes after the microspheres are activated.

[0009] Preferably, the activating agents used in the activation are 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

[0010] Preferably, the purity of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is ≥98%; the purity of the N-hydroxysuccinimide is ≥98%.

[0011] Preferably, the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 2-3:4-6.

[0012] Preferably, the method for activating the microspheres is: mixing the microsphere solution with an activator, and activating for 10 to 60 min at a pH of 5.5 to 6.5, a temperature of 20 to 30° C., and a speed of 500 to 1500 rrpm.

[0013] Preferably, the concentration of the microsphere solution is 0.4-0.6 mg / mL; the concentration of the activator in the solution after mixing with the activator is 2-5 mg / mL.

[0014] Preferably, the bioactive substance comprises one of an antibody, an antigen, an oligonucleotide, a cytokine, a polypeptide, a protein complex and a biomarker.

[0015] Preferably, the coupling temperature is 20-30° C., the speed is 15-20 rpm, and the time is 1.5-4 h.

[0016] The inventors found that the reaction kinetics and stability of the activator and the carboxyl group on the surface of the microspheres have an important influence on the coupling effect, and the stability of the intermediate after activation and its coupling reaction rate with the biological molecule are the key factors affecting the labeling effect. Based on the above findings, the inventors effectively reduced the batch difference and false positive results by adjusting the addition time of the bioactive substance, and improved the specificity and batch consistency of the immunoassay.

[0017] The present invention does not add the bioactive substance immediately for coupling after the microsphere activation reaction is completed, but sets an appropriate delay time, during which the unstable intermediate is allowed to hydrolyze or react with other molecules, reducing the chance of non-specific binding with the coupling object, improving the reaction environment on the surface of the microsphere, reducing non-specific adsorption, and at the same time, the stability of the activated intermediate is enhanced, which can ensure that the activated intermediate participates in the reaction in the best state, avoiding the side reaction and impurity generation caused by adding too early or too late, thereby ensuring the high purity and high yield of the product. When the delay time is over, the biomolecule is added for coupling reaction, at which time the reaction activity of the intermediate is more stable, and the coupling reaction with the biomolecule is more controllable, thereby helping to reduce the occurrence of false positive phenomena caused by batch differences and non-specific binding.

[0018] The present invention does not need to introduce additional reagents or steps, and can achieve the reduction of batch differences and false positives by optimizing the addition time of the coupling object. It is simple to operate and easy to control, which is conducive to reducing false positive results caused by nonspecific binding, making the biocoupling reaction more sensitive and reliable, and thus can improve the detection sensitivity, specificity and accuracy. It has extremely high practical value for fields that require mass production and have high requirements for product quality. The method of the present invention can provide a more reliable and stable tool for biomedical research and clinical diagnosis, and has good application prospects. DETAILED DESCRIPTION

[0019] The present invention provides a method for controlling batch differences and improving specificity by changing the time of adding raw materials after microsphere activation, wherein biologically active substances are added for coupling after a certain period of time after microsphere activation.

[0020] In the present invention, the microspheres include carboxyl-modified microspheres.

[0021] In the present invention, the bioactive substance is added for coupling 18 to 30 minutes after the microspheres are activated.

[0022] In the present invention, the particle size of the carboxyl-modified microspheres is 100 nm to 10 μm.

[0023] In the present invention, the activating agents used in the activation are 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

[0024] In the present invention, the purity of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is ≥98%; the purity of the N-hydroxysuccinimide is ≥98%.

[0025] In the present invention, the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 2-3:4-6, preferably 2-3:4.5-5.5, and more preferably 2.5:5.

[0026] In the present invention, the method for activating the microspheres is: mixing the microsphere solution with an activator, and activating for 10 to 60 min at a pH of 5.5 to 6.5, a temperature of 20 to 30° C., and a speed of 600 to 1500 rpm.

[0027] In the present invention, the method for activating the microspheres is preferably: mixing the microsphere solution with the activator, and activating for 20 to 50 min at a pH of 5.8 to 6.2, a temperature of 22 to 28° C., and a speed of 500 to 1500 rpm; further preferably: mixing the microsphere solution with the activator, and activating for 40 min at a pH of 6.0, a temperature of 25° C., and a speed of 1000 rpm.

[0028] In the present invention, the concentration of the microsphere solution is 0.4-0.6 mg / mL, preferably 0.45-0.55 mg / mL, and more preferably 0.5 mg / mL; the concentration of the activator in the solution after mixing with the activator is 2-5 mg / mL, preferably 2-4 mg / mL, and more preferably 3 mg / mL.

[0029] In the present invention, after the microspheres are activated, they are cleaned with a microsphere cleaning solution, and the cleaned microspheres are dissolved in an activation coupling buffer and allowed to stand at a temperature of 20-30°C for 20-30 min, preferably at a temperature of 22-28°C for 22-28 min, more preferably at a temperature of 24-26°C for 24-26 min, and more preferably at a temperature of 25°C for 25 min.

[0030] In the present invention, the microsphere cleaning solution is a phosphate buffer with a pH of 7.4; the activated coupling buffer is a MES buffer with a pH of 6.0 and a concentration of 100 mmol / L; and the volume ratio of the microsphere to the activated coupling buffer is 1:10.

[0031] In the present invention, the bioactive substance includes one of an antibody, an antigen, an oligonucleotide, a cytokine, a polypeptide, a protein complex and a biomarker.

[0032] In the present invention, the antibodies include monoclonal / polyclonal antibodies, chimeric / humanized antibodies and antibody fragments.

[0033] In the present invention, the antigens include natural antigens and recombinant antigens.

[0034] In the present invention, the oligonucleotides include DNA / RNA probes, primers and adaptors.

[0035] In the present invention, during coupling, the bioactive substance is added to the activated coupling buffer containing the microspheres, and the concentration of the bioactive substance is controlled to be 10-30 μg / mL.

[0036] In the present invention, the coupling temperature is 20-30°C, preferably 22-28°C, more preferably 24-26°C, and more preferably 25°C; the speed is 15-20rpm, preferably 16-19rpm, and more preferably 18rpm; the time is 1.5-4h, preferably 1.8-3h, and more preferably 2h.

[0037] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0038] The carboxyl-modified microspheres, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) used in the following examples were from Thermo Scientific; MES (2-(N-morpholino)ethanesulfonic acid) was from Baker Biotechnology Co., Ltd.; house dust mite extract (HDM, D. pteronyssinus, hereinafter referred to as D1 raw material, product number XPB82D3A2.5) and egg white (egg white, chicken, hereinafter referred to as F1 raw material, product number XPF272D3A2.5) were from Greer labs.

[0039] Example 1

[0040] A method for controlling batch differences and improving specificity by changing the time of adding raw materials after microsphere activation, the steps are as follows: The carboxyl-modified microspheres with an average particle size of 1 μm were washed twice with a phosphate buffer solution at pH 7.4 to remove impurities and residues. The washed carboxyl-modified microspheres were dispersed in a MES buffer solution at pH 6.0 and a concentration of 100 mmol / L to make the concentration of the carboxyl-modified microspheres 0.5 mg / mL. NHS was then added to make the final concentration of NHS 5 mg / mL, and EDC was then added to make the final concentration of EDC 2.5 mg / mL. The microspheres were activated in a shaker at pH 6.0, room temperature (25°C), and 1000 rpm for 40 min to obtain activated carboxyl-modified microspheres.

[0041] The activated carboxyl-modified microspheres were washed twice with phosphate buffer at pH 7.4, and the washed carboxyl-modified microspheres were dissolved in MES buffer at pH 6.0 and concentration of 100 mmol / L, and the volume ratio of carboxyl-modified microspheres to MES buffer was 1:10. The dissolved carboxyl-modified microsphere solution was divided into 4 groups, and the D1 raw material was added after standing at room temperature (25°C) for 10, 20, 30 and 40 min, respectively, to make the concentration of D1 raw material 1 mg / mL, and continued to react at room temperature (25°C) and 18 rpm (using a four-dimensional rotating mixer) for 2 h to fully couple the D1 raw material with the carboxyl-modified microspheres. The coupled microspheres were washed twice with PBS buffer to remove the unbound D1 raw material, and 50 mmol / L phosphate buffer containing 1% (mass concentration) bovine serum albumin and pH 7.4 was added for blocking, and incubated at room temperature (25°C) for 2 h. After blocking, it was stored in the dark.

[0042] Example 2

[0043] The difference between this example and example 1 is that in this example, the dissolved carboxyl-modified microsphere solution is divided into 4 groups, and the D1 raw material is added after standing at room temperature (25° C.) for 25, 30, 35 and 40 minutes respectively, and the remaining steps are the same as in example 1.

[0044] Example 3

[0045] The difference between this example and Example 1 is that in this example, the dissolved carboxyl-modified microsphere solution is divided into 4 groups, and the D1 raw material is added after standing at room temperature (25° C.) for 25 minutes. The remaining steps are the same as in Example 1.

[0046] Example 4

[0047] The difference between this example and example 1 is that in this example, the dissolved carboxyl-modified microsphere solution is divided into 4 groups, and the D1 raw material is added after standing at room temperature (25° C.) for 10, 15, 45 and 55 minutes respectively, and the remaining steps are the same as in example 1.

[0048] Example 5

[0049] The purpose of this example is to evaluate the coupling effect of the coupled microspheres obtained in Examples 1 to 4.

[0050] 1. False positive analysis: Serum samples without D1-specific antibodies were used as blank samples to evaluate the false positive values ​​of the coupled microspheres of the blank samples tested by the chemiluminescence instrument in each embodiment (luminescence signal values ​​≥ 1000 were false positive values). No false positives indicate better specificity and less nonspecific binding.

[0051] 2. Performance analysis: Analyze the luminescent signal value of each group of the same sample. A higher luminescent signal value usually indicates a higher binding efficiency between the antigen and the target coupling object, and a better coupling effect.

[0052] 3. Relative deviation between batches (R): Calculate the mean of 3 measurements for each batch for each group, and calculate the relative deviation between batches. The smaller the R value, the better the stability between different batches and the more consistent the coupling effect. The calculation method is as follows:

[0053]

[0054] Where: represents the mean of 3 measurements per batch (i = 1, 2, 3, 4); express The maximum value in ; express The minimum value in ; Represents the batch reagent test mean.

[0055] The evaluation results of the coupling effects of the coupled microspheres prepared in each embodiment are shown in Tables 1 to 4.

[0056] Table 1 Coupling effect of coupled microspheres prepared in Example 1

[0057] Table 2 Coupling effect of coupled microspheres prepared in Example 2

[0058] Table 3 Coupling effect of coupled microspheres prepared in Example 3

[0059] Table 4 Coupling effect of coupled microspheres prepared in Example 4

[0060] It can be seen from Table 1 that the groups in which the D1 raw material was added within 20 minutes after activation were all false positive; further, according to the measurement results of Table 2, it can be confirmed that the optimal addition time of the D1 raw material is 25 minutes; it can be seen from the measurement results of Table 3 that the inter-batch deviations of Example 3 are all less than 10%, that is, the inter-batch differences between different batches are small. It can be seen from Table 4 that the time of adding the raw material after activation is randomly distributed, and the measured values ​​are unstable.

[0061] Example 6

[0062] The difference between this embodiment and embodiment 1 is that the coupling raw material used in this embodiment is F1 raw material, and the remaining steps are the same as those in embodiment 1.

[0063] Example 7

[0064] The difference between this example and Example 6 is that in this example, the dissolved carboxyl-modified microsphere solution is divided into 4 groups, and the F1 raw material is added after standing at room temperature (25° C.) for 15, 25, 30 and 35 minutes respectively, and the remaining steps are the same as Example 6.

[0065] Example 8

[0066] The difference between this example and Example 6 is that in this example, the dissolved carboxyl-modified microsphere solution is divided into 4 groups, and the F1 raw material is added after standing at room temperature (25° C.) for 20 minutes. The remaining steps are the same as in Example 6.

[0067] Example 9

[0068] The difference between this example and Example 6 is that in this example, the dissolved carboxyl-modified microsphere solution is divided into 4 groups, and the F1 raw material is added after standing at room temperature (25° C.) for 10, 20, 30 and 35 minutes respectively, and the remaining steps are the same as Example 6.

[0069] Example 10

[0070] The purpose of this example is to evaluate the coupling effect of the coupled microspheres obtained in Examples 6 to 9. The evaluation method is the same as that in Example 5, and the evaluation results are shown in Tables 5 to 8.

[0071] Table 5 Coupling effect of coupled microspheres prepared in Example 6

[0072] Table 6 Coupling effect of coupled microspheres prepared in Example 7

[0073] Table 7 Coupling effect of coupled microspheres prepared in Example 8

[0074] Table 8 Coupling effect of coupled microspheres prepared in Example 9

[0075] It can be seen from Table 5 that the groups in which the time of adding F1 raw material after activation is within 10 minutes are all false positive; according to the measurement results of Table 6, it is further confirmed that the optimal time of adding F1 raw material is 20 minutes; from the measurement results of Table 7, it can be seen that the inter-batch deviation of Example 3 is less than 10%, that is, the inter-batch difference between different batches is small. It can be seen from Table 8 that the time of adding raw materials after activation is randomly distributed, and the measured value is unstable.

[0076] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for controlling batch differences and improving specificity by changing the time of adding raw materials after microsphere activation, characterized in that: After a certain period of time after the microspheres are activated, biologically active substances are added for coupling.

2. The method according to claim 1, characterized in that The microspheres include carboxyl-modified microspheres.

3. The method according to claim 2, characterized in that The bioactive substances were added for coupling 18 to 30 min after the microspheres were activated.

4. The method according to claim 3, characterized in that The activating agents used in the activation are 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

5. The method according to claim 4, characterized in that The purity of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is ≥98%; the purity of the N-hydroxysuccinimide is ≥98%.

6. The method according to claim 5, characterized in that The mass ratio of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 2-3:4-6.

7. The method according to claim 6, characterized in that The microsphere activation method comprises: mixing the microsphere solution with the activator, and activating the mixture for 10 to 60 minutes at a pH of 5.5 to 6.5, a temperature of 20 to 30° C., and a speed of 500 to 1500 rpm.

8. The method according to claim 7, characterized in that The concentration of the microsphere solution is 0.4-0.6 mg / mL; the concentration of the activator in the solution after mixing with the activator is 2-5 mg / mL.

9. The method according to claim 8, characterized in that The bioactive substance includes one of an antibody, an antigen, an oligonucleotide, a cytokine, a polypeptide, a protein complex and a biomarker.

10. The method according to any one of claims 1 to 9, characterized in that: The coupling temperature is 20-30° C., the speed is 15-20 rpm, and the time is 1.5-4 h.

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