Blocking agent as well as preparation method and application thereof

Abstract

The invention relates to the technical field of immunological detection, in particular to a blocking agent and a preparation method and application thereof. According to the blocking agent and the preparation method and application thereof, BSA and 3, 5-diiodo tyrosine serve as raw materials of the blocking agent, and due to the fact that the structure of 3, 5-diiodo tyrosine is similar to that of T4, the prepared blocking agent can effectively eliminate non-specific adsorption in T4 antibody detection, and therefore the accuracy of a detection result is improved. Experiments prove that in a detection reaction, a detection result of a sample added with the blocking agent is consistent with a Rogowski detection result, the blocking effect is the best when the added blocking agent is 20-40 [mu] g / mL, and meanwhile, the detection specificity of a normal sample is not influenced.

Description

Technical Field

[0001] The present invention relates to the technical field of immunological detection, and in particular to a blocking agent and a preparation method and application thereof. Background Art

[0002] Thyroxin (Thyroxin(e), Thx, T4) is a hormone synthesized and secreted by thyroid follicular cells. It is released into the blood circulation in a free form and quickly binds to plasma proteins. Thyroxine, also known as tetraiodothyronine, is an iodinated tyrosine derivative formed by coupling two 3,5-diiodotyrosine molecules and is the main hormone of the thyroid gland. There are three configurations: DL, L, and D. T4 can promote the absorption of sugar by the small intestine, promote the decomposition of glycogen into glucose, and increase blood sugar concentration. Patients with hyperthyroidism (commonly known as "hyperthyroidism") have excessive secretion of thyroid hormone and vigorous functional metabolism, which accelerates the oxidation and decomposition of energy substances in the body and releases too much heat. Quantitative determination of human serum fT4 is of great significance for the diagnosis of thyroid diseases and the pathological and physiological research of the thyroid gland.

[0003] The horseradish enzyme system magnetic microparticle chemiluminescence detection method generally uses a competitive method to detect free antigens in serum. The competitive method mode requires the use of coated antigens and labeled antibodies. T4 antigen is a small molecule, and the effect of directly coating it on magnetic beads is poor. The existing technology generally couples T4 to BSA and then coats it on magnetic beads. In the detection process using this mode, there will be a phenomenon that the results are inconsistent with the Roche test results. Some serum may have non-specific adsorption, and the accuracy of the test results cannot be guaranteed. Summary of the invention

[0004] The invention provides a blocking agent and a preparation method and application thereof. The blocking agent prepared by the invention can eliminate nonspecific adsorption in T4 antibody detection, thereby improving the accuracy of the detection result.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a blocking agent, which is formed by cross-linking BSA and 3,5-diiodotyrosine in a molar ratio of 1:(5-10).

[0007] The present invention utilizes the principle that 3,5-diiodotyrosine is similar to T4 in structure to prepare a blocker. Compared with other T4 structural analogs, the blocker prepared by 3,5-diiodotyrosine and BSA can better eliminate nonspecific adsorption in T4 antibody detection, thereby improving the accuracy of the detection result.

[0008] In some embodiments, it is formed by cross-linking BSA and 3,5-diiodotyrosine at a molar ratio of 1:5; or

[0009] It is formed by cross-linking BSA and 3,5-diiodotyrosine in a molar ratio of 1:8; or

[0010] It is formed by cross-linking BSA and 3,5-diiodotyrosine in a molar ratio of 1:10.

[0011] The present invention also provides a method for preparing the blocking agent, wherein the 3,5-diiodotyrosine and the activated BSA are mixed, cross-linked and terminated to obtain the blocking agent.

[0012] In some embodiments, the activation comprises dissolving the BSA in MES buffer and mixing the mixture with the activation reagent to obtain the activated BSA.

[0013] In some embodiments, the activated reagent includes 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, the molar ratio of the BSA, the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and the N-hydroxysuccinimide is 1:(100~200):(200~400), the mixing reaction time is 0.5~1h, and the mixing reaction temperature is 25~30°C.

[0014] In some specific embodiments, the pH of the MES buffer is 4.7, the molar ratio of the BSA, the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and the N-hydroxysuccinimide is 1:100:200, 1:150:300 or 1:200:300, the mixing reaction time is 1 hour, and the mixing reaction temperature is 25°C.

[0015] In some embodiments, the cross-linking reaction time is 3-5 hours, the cross-linking reaction temperature is 25-30° C., and the cross-linking reaction pH is 7-9.

[0016] In some specific embodiments, the cross-linking reaction time is 3h, 4h or 5h, the cross-linking reaction temperature is 25°C, and the cross-linking reaction pH is 7, 8 or 9.

[0017] In some embodiments, the reagent for terminating the reaction includes at least one of Tris, ethanolamine, glycine, and lysine, the molar ratio of BSA to the reagent for terminating the reaction is 1:(20-50), and the time for the termination reaction is 1-3 hours.

[0018] In some specific embodiments, the reagent for terminating the reaction is Tris, the molar ratio of BSA to the reagent for terminating the reaction is 1:40, and the time for terminating the reaction is 2 hours.

[0019] In some embodiments, after the termination reaction, the method further comprises using a molecular sieve to remove unreacted small molecules in the reaction solution of the termination reaction to obtain the blocking agent.

[0020] In some specific embodiments, the molecular sieve is a G25 desalting column.

[0021] The present invention provides application of the blocker or the blocker prepared by the preparation method in preparing a kit for detecting T4 antibodies.

[0022] The present invention provides a kit for detecting T4 antibodies, comprising the blocking agent or the blocking agent prepared by the preparation method.

[0023] The present invention provides a blocker and a preparation method and application thereof. The blocker provided by the present invention uses BSA and 3,5-diiodotyrosine as raw materials. Since 3,5-diiodotyrosine is similar to T4 in structure, the prepared blocker can effectively eliminate nonspecific adsorption in T4 antibody detection, thereby improving the accuracy of the detection result. It has been verified by experiments that in the detection reaction, the detection result of the sample with the added blocker is consistent with the Roche detection result, and the blocking effect is best when the added blocking dose is 20-40ug / mL, and at the same time, it has no effect on the detection specificity of normal samples. DETAILED DESCRIPTION

[0024] The present invention provides a blocking agent and a preparation method and application thereof. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0025] The test materials used in the present invention are all common commercial products and can be purchased in the market. The present invention is further described below in conjunction with the embodiments.

[0026] Example 1

[0027] A method for preparing a blocking agent in a T4 antibody test is as follows:

[0028] The first step is BSA activation

[0029] BSA was dissolved in MES PH4.7 to a concentration of 1 mg / mL, and EDC / NHS was dissolved in MES PH4.7, and mixed in a molar ratio of nBSA:EDC:NHS=1:100:200, and reacted at 25°C for 1 h.

[0030] Step 2: Cross-linking reaction

[0031] 3,5-diiodotyrosine (structure shown below) was dissolved into 1 mg / mL with ultrapure water, and ultrasonic dissolution was performed. The activated BSA and 3,5-diiodotyrosine were mixed in a molar ratio of 1:5. After mixing, the pH was adjusted to 7 with carbonate buffer, and the reaction was carried out at 25°C for 5 hours. After the reaction, the terminator Tris was added to terminate the reaction (40 moles of terminator were added per mole of BSA) for 2 hours. The sample was passed through a G25 desalting column to remove excess small molecules, and the sample was concentrated to 5 mg / mL.

[0032]

[0033] 3,5-Diiodotyrosine

[0034] Step 3: Post-processing and saving

[0035] Accurately measure the volume of 3,5-diiodotyrosine-BSA, add an equal volume of glycerol, mix well, measure the concentration, and store at -20°C away from light.

[0036] Example 2

[0037] A method for preparing a blocking agent in a T4 antibody test is as follows:

[0038] The first step is BSA activation

[0039] BSA was dissolved in MES PH4.7 to a concentration of 5 mg / mL, and EDC / NHS was dissolved in MES PH4.7. The mixture was mixed in a molar ratio of nBSA:EDC:NHS=1:150:300 and reacted at 25°C for 0.7 h.

[0040] Step 2: Cross-linking reaction

[0041] 3,5-diiodotyrosine was dissolved into 1 mg / mL with ultrapure water and sonicated, and the activated BSA and 3,5-diiodotyrosine were mixed in a molar ratio of 1:8. After mixing, the pH was adjusted to 8 with carbonate buffer and reacted at 25°C for 4 hours. After the reaction, the terminator Tris was added to terminate the reaction (40 moles of terminator were added per mole of BSA) for 2 hours. The sample was desalted with a G25 desalting column to remove excess small molecules and the sample was concentrated to 8 mg / mL.

[0042] Step 3: Post-processing and saving

[0043] Accurately measure the volume of 3,5-diiodotyrosine-BSA, add an equal volume of glycerol, mix well, measure the concentration, and store at -20°C away from light.

[0044] Example 3

[0045] A method for preparing a blocking agent in a T4 antibody test is as follows:

[0046] The first step is BSA activation

[0047] BSA was dissolved in MES PH4.7 to a concentration of 10 mg / mL, and EDC / NHS was dissolved in MES PH4.7, and mixed in a molar ratio of nBSA:EDC:NHS=1:200:400, and reacted at 25°C for 0.5 h.

[0048] Step 2: Cross-linking reaction

[0049] 3,5-diiodotyrosine was dissolved into 1 mg / mL with ultrapure water and sonicated. The activated BSA and 3,5-diiodotyrosine were mixed in a molar ratio of 1:10. After mixing, the pH was adjusted to 9 with carbonate buffer and reacted at 25°C for 3 hours. After the reaction, the terminator Tris was added to terminate the reaction (40 moles of terminator were added per mole of BSA) for 2 hours. The sample was passed through a G25 desalting column to remove excess small molecules and the sample was concentrated to 10 mg / mL.

[0050] Step 3: Post-processing and saving

[0051] Accurately measure the volume of 3,5-diiodotyrosine-BSA, add an equal volume of glycerol, mix well, measure the concentration, and store at -20°C away from light.

[0052] Comparative Example 1

[0053] Tyrosine, 3-iodotyrosine, phenylalanine, and 4-methoxyphenylalanine were selected as cross-linking agents to replace 3,5-diiodotyrosine in Example 1. The remaining steps and parameters were the same as in Example 1 to prepare the corresponding blocking agents, wherein the structural formulas of the four reagents are as follows.

[0054]

[0055] Tyrosine

[0056]

[0057] 3-Iodotyrosine

[0058]

[0059] Phenylalanine

[0060]

[0061] 4-Methoxyphenylalanine

[0062] Effect Example 1 Test Example Performance Test of Blocker

[0063] The blockers prepared in Examples 1-3 and Comparative Example 1 were used for sample detection, and the luminescence value of the calibration product was detected in the horseradish enzyme system magnetic microparticle chemiluminescence system. The blocker was added to the magnetic bead suspension coated with T4-BSA antigen, and the addition amount was 20ul / mL magnetic beads, mixed and set aside; the magnetic beads were washed after binding with the sample, and the corresponding enzyme-labeled antibody was added. The benchmark selected the corresponding kit product of Roche, and 5 samples that were different from the Roche test were selected for screening and detection of blockers. The results are shown in Table 1 below. The range of fT4 in serum is 10-22pmol / L. After adding the blocker, when the measured value is within the range, it indicates a blocking effect, otherwise there is no blocking effect. It can be seen that the embodiment has a blocking effect on all 5 samples, and the blocking effect of the remaining blockers is poor.

[0064] Table 1

[0065]

[0066] Effect Example 2 Testing of Blocker Addition and Its Impact on Routine Sample Testing

[0067] The blocker prepared in Example 1 was added to the magnetic beads in different dosages (5-80ug / mL) and mixed for later use. The detection method was the same as that in Example 1. Five samples that were different from the Roche test were selected for dosage detection, and 10 samples that were not different from the Roche test were selected for specificity detection. The results are shown in Tables 2 and 3. The results show that the blocking effect is best when the blocking dose is 20-40ug / mL, and it has no effect on the detection specificity of normal samples.

[0068] Table 2

[0069] Table 3

[0070] Effect Example 3: Test whether the coating blocker has an effect on the detection

[0071] The coated magnetic beads were prepared by replacing the coated antigen with the blocker prepared in Examples 1-3, with a coating amount of 2 ug / mL, and the above 5 samples were directly detected in the horseradish enzyme system magnetic microparticle chemiluminescence system without adding a blocker, and the results are shown in Table 4. The detection values ​​were all less than 0.1, indicating that the blocker prepared in the example had no effect on the T4 antibody detection.

[0072] Table 4

[0073]

[0074] The above are only preferred embodiments 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 principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A blocking agent, characterized in that It is formed by cross-linking BSA and 3,5-diiodotyrosine in a molar ratio of 1:(5~10).

2. The blocking agent according to claim 1, characterized in that It is formed by cross-linking BSA and 3,5-diiodotyrosine in a molar ratio of 1:5; or It is formed by cross-linking BSA and 3,5-diiodotyrosine in a molar ratio of 1:8; or It is formed by cross-linking BSA and 3,5-diiodotyrosine in a molar ratio of 1:

10.

3. The method for preparing the blocking agent according to claim 1 or 2, characterized in that: The blocking agent is obtained by mixing the 3,5-diiodotyrosine and the activated BSA, performing cross-linking reaction and terminating the reaction.

4. The preparation method according to claim 3, characterized in that: The activation comprises dissolving the BSA in MES buffer and mixing it with the activation reagent to obtain the activated BSA.

5. The preparation method according to claim 4, characterized in that: The activated reagent includes 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, the molar ratio of the BSA, the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and the N-hydroxysuccinimide is 1:(100-200):(200-400), the mixing reaction time is 0.5-1h, and the mixing reaction temperature is 25-30°C.

6. The preparation method according to any one of claims 3 to 5, characterized in that The cross-linking reaction time is 3-5 hours, the cross-linking reaction temperature is 25-30° C., and the cross-linking reaction pH is 7-9.

7. The preparation method according to any one of claims 3 to 6, characterized in that The reagent for terminating the reaction includes at least one of Tris, ethanolamine, glycine, and lysine, the molar ratio of the BSA to the reagent for terminating the reaction is 1:(20-50), and the time for the terminating reaction is 1-3 hours.

8. The preparation method according to any one of claims 3 to 7, characterized in that After the termination reaction, the method further comprises using a molecular sieve to remove unreacted small molecules in the reaction solution of the termination reaction to obtain the blocking agent.

9. Use of the blocking agent according to claim 1 or 2 or the blocking agent prepared by the preparation method according to any one of claims 3 to 8 in preparing a kit for detecting T4 antibodies.

10. A kit for detecting T4 antibodies, characterized in that: The invention comprises the blocking agent according to claim 1 or 2 or the blocking agent prepared by the preparation method according to any one of claims 3 to 8.

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