A dissociation agent, its preparation method and uses

Abstract

This invention discloses a dissociation agent, its preparation method, and its applications. The dissociation agent comprises a chelating agent, a competitive binding agent, an inclusion agent, a denaturing agent, a surfactant system, a stabilizer, and a buffer system. The competitive binding agent is salicylic acid, the inclusion agent is hydroxypropyl-β-cyclodextrin, and the surfactant system is a compound system of the nonionic surfactant Brij-35 and the amphoteric surfactant 3-sulfopropyltetradecyl dimethyl betaine. This invention efficiently dissociates bound hormones through multiple synergistic effects of chelation, competition, inclusion, and denaturation; the components are mild, have good compatibility with chemiluminescence detection systems, and do not interfere with the detection signal; simultaneously, the addition of stabilizers and preservatives significantly improves the long-term stability of the reagent. This invention exhibits high dissociation efficiency, strong stability, and simple operation, with a correlation of over 0.99 with imported reagents, demonstrating promising clinical application prospects and industrialization value.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, specifically relating to a dissociation agent, its preparation method, and its uses. Background Technology

[0002] Triiodothyronine (T3) and thyroxine (T4) are the main hormones secreted by the thyroid gland. Accurate measurement of their total serum levels (TT3, TT4) is crucial for the diagnosis and monitoring of treatment efficacy in diseases such as hyperthyroidism and hypothyroidism. In human blood circulation, over 99% of T3 and T4 exist in a reversibly bound state, primarily bound to transport proteins such as thyroid-binding globulin (TBG), thyroid-binding prealbumin (TBPA), and albumin. Therefore, before immunoassay, a dissociating agent must be used to fully and stably release the bound hormones; the dissociation efficiency directly determines the accuracy and repeatability of the test results. Currently, several dissociating agents for TT3 and TT4 detection have been disclosed.

[0003] Patent CN117169519B discloses a dissociation agent comprising elastase, a disulfide bond reducing agent, and a nonionic surfactant. This method utilizes protease to enzymatically hydrolyze bound proteins, but enzyme reagents have stringent requirements for storage and reaction conditions, are costly, and may exhibit batch-to-batch variability.

[0004] Patent CN112362432B discloses a dissociation agent applicable to TT3 and TT4, comprising a metal chelate, an N-heterocyclic compound (such as 5-bromo-2-methylpyridine), a surfactant, and an auxiliary agent (such as ammonium 8-phenylamino-1-naphthalenesulfonate). While this approach exhibits good dissociation performance, the ammonium 8-phenylamino-1-naphthalenesulfonate (ANS) itself is fluorescent, which may cause background interference in some optical detection systems.

[0005] Furthermore, patent CN121027541A discloses a dissociation agent for fluorescence immunochromatography, comprising acetate-sodium acetate buffer, disodium ethylenediaminetetraacetate, perfluoroalkyl ethyl methacrylate, and Tween-20. This solution aims to address the interference of traditional dissociation agents (ANS) on rare-earth europium, but it is only applicable to T3 and lacks universality. Moreover, its system is acidic (pH 5.5±1), which may not be fully compatible with the optimal pH environment of subsequent chemiluminescence detection systems. Furthermore, perfluoroalkyl ethyl methacrylate poses environmental risks, and its application in in vitro diagnostic reagent formulations requires careful consideration of safety and environmental protection.

[0006] In summary, existing dissociation agents still have some technical shortcomings, such as: 1) some components (such as ANS) may interfere with the detection signal; 2) enzyme components have poor stability and high cost; 3) the dissociation system is not mild enough and may quench or inhibit antibodies or markers (such as acridinium ester and alkaline phosphatase); 4) the dissociation of abnormal samples (such as hemolysis and lipemia) is not thorough enough, affecting the precision and accuracy of detection.

[0007] Therefore, developing a novel dissociation agent that is efficient, mild, stable, and highly compatible with chemiluminescence systems remains a pressing technical problem to be solved in this field. Summary of the Invention

[0008] The present invention aims to provide a novel dissociation agent with high dissociation efficiency, good compatibility with chemiluminescence systems, strong stability, and good anti-interference ability against abnormal samples, as well as its preparation method and uses.

[0009] The technical solution adopted in this invention is as follows: A dissociation agent comprising a chelating agent, a competitive binding agent, an inclusion agent, a denaturing agent, a surfactant system, a buffer system, and a stabilizer; The chelating agent is DTPA; the competitive chelating agent is salicylic acid; the inclusion agent is hydroxypropyl-β-cyclodextrin; and the denaturing agent is guanidine hydrochloride.

[0010] In one embodiment, the surfactant system is a compound system of nonionic surfactant and amphoteric surfactant.

[0011] In one embodiment, the nonionic surfactant is Brij-35, and the amphoteric surfactant is 3-sulfopropyltetradecyl dimethyl betaine.

[0012] In one embodiment, the buffer system is a 10mM~50mM HEPES buffer solution; the stabilizer is mannitol.

[0013] Optionally, the dissociation agent further comprises a preservative, wherein the preservative is ProClin 300.

[0014] In one embodiment, the pH of the dissociation agent is 7.0 to 7.5.

[0015] In one embodiment, the dissociation agent, per 100 mL, comprises the following components: DTPA 0.1 g to 0.2 g, salicylic acid 0.01 g to 0.1 g, hydroxypropyl-β-cyclodextrin 0.2 g to 1.0 g, Brij-35 0.05 mL to 0.2 mL, guanidine hydrochloride 1.0 g to 2.0 g, 3-sulfopropyltetradecyl dimethyl betaine 0.01 g to 0.1 g, stabilizer 0.5 g to 2.0 g, preservative 0.01 mL to 0.1 mL, with the balance being water.

[0016] In one embodiment, the dissociation agent, per 100 mL, comprises the following components: DTPA 0.15 g, salicylic acid 0.05 g, hydroxypropyl-β-cyclodextrin 0.5 g, Brij-35 0.1 mL, guanidine hydrochloride 1.5 g, 3-sulfopropyltetradecyl dimethyl betaine 0.05 g, stabilizer 1 g, preservative 0.05 mL, with the remainder being water; the pH of the dissociation agent is 7.2~7.4.

[0017] The present invention also provides a method for a dissociating agent as described in any of the foregoing claims, comprising the following steps: 1) Provide a buffer solution, add salicylic acid, and adjust the pH to 7.0~7.5; 2) Add hydroxypropyl-β-cyclodextrin, DTPA, guanidine hydrochloride, amphoteric surfactant, and stabilizer sequentially to the solution obtained in step 1); stir thoroughly to dissolve each component before adding it. 3) Add a nonionic surfactant to the solution obtained in step 2) and mix well; 4) Make up the volume, add preservative, filter, and the product is ready.

[0018] The present invention also provides a kit comprising the dissociation agent described in any one of the above-mentioned claims.

[0019] The present invention further provides the use of the dissociating agent as described in any of the above descriptions, namely, using the dissociating agent to prepare a kit for detecting one or both of total triiodothyronine and total thyroxine in serum.

[0020] Compared with the prior art, the present invention has the following significant advantages: 1. The dissociation agent provided by this invention achieves deep, efficient, and accurate dissociation of bound TT3 / TT4 through the synergistic effects of multiple chelating agents (DTPA), denaturing agents (guanidine hydrochloride), competitive binding agents (salicylic acid), and inclusion solubilizers (hydroxypropyl-β-cyclodextrin).

[0021] 2. All components selected in this invention are mild reagents, avoiding the use of strong oxidants, and do not contain self-fluorescent substances such as ANS. They have no quenching or inhibitory effect on active substances such as labeled antibodies, acridinium esters, and enzymes in the chemiluminescent immunoassay system. The system has excellent compatibility, does not interfere with the detection signal, and ensures the stability and sensitivity of the detection signal.

[0022] 3. This invention is a completely chemical, non-enzymatic system, which avoids the risks of low-temperature storage and transportation, inactivation, and high costs associated with biological enzyme preparations. It is simple to prepare, has good repeatability, and is more easily adapted to fully automated chemiluminescence detection platforms, thus having good prospects for industrialization.

[0023] 4. This invention significantly improves the long-term storage stability and thermal stability of the deionizing agent by further adding stabilizer (mannitol) and broad-spectrum preservative (ProClin 300), resulting in a longer shelf life. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be further described below in conjunction with specific embodiments. However, it should be understood that these embodiments are only for explaining this invention and are not intended to limit the scope of protection of this invention. All modifications, equivalent substitutions, or improvements made based on the technical solutions of this invention should be included within the scope of protection of this invention.

[0025] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0026] DTPA: Diethylenetriaminepentaacetic acid, CAS number is 67-43-6.

[0027] Brij-35: Lauryl alcohol polyoxyethylene ether, CAS number is 9002-92-0.

[0028] Salicylic acid: o-hydroxybenzoic acid, CAS number is 69-72-7.

[0029] Hydroxypropyl-β-cyclodextrin, CAS number is 128446-35-5.

[0030] Guanidine hydrochloride, CAS number is 50-01-1.

[0031] 3-Sulfopropyltetradecyldimethylbetaine: CAS number is 14933-09-6.

[0032] Mannitol, CAS number 87-78-5.

[0033] HEPES: N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, CAS number is 7365-45-9.

[0034] Example 1 A dissociation agent, with the following formulation: The dissociation agent, per 100 mL, consists of the following components: DTPA 0.15 g, salicylic acid 0.05 g, hydroxypropyl-β-cyclodextrin 0.5 g, Brij-35 0.1 mL, guanidine hydrochloride 1.5 g, 3-sulfopropyltetradecyl dimethyl betaine 0.05 g, mannitol 1 g, ProClin 300 0.05 mL, with the remainder being water. Adjust the pH to 7.2–7.4.

[0035] The preparation method is as follows: 1) Measure 80% of the total volume of purified water and add it to the reactor. Add HEPES to bring the final concentration to 20 mM and stir until completely dissolved. Add salicylic acid and continue stirring until completely dissolved; adjust the pH to 7.2–7.4 using 0.1 M NaOH solution and set aside.

[0036] 2) Add hydroxypropyl-β-cyclodextrin, DTPA, guanidine hydrochloride, 3-sulfopropyltetradecyl dimethyl betaine, and mannitol in sequence. Stir until completely dissolved after each addition before adding the next component.

[0037] 3) Slowly add Brij-35 to the solution obtained in step 2) and stir at low speed until the solution is uniform and transparent.

[0038] 4) Adjust the volume to the final volume with purified water and stir until well mixed. Add ProClin 300 and continue stirring until completely mixed. Filter using a 0.22μm filter membrane and collect the filtrate.

[0039] The prepared dissociation agent was sealed and stored at 2–8°C for later use.

[0040] Example 2 The dissociation agent, per 100 mL, consists of the following components: DTPA 0.1 g, salicylic acid 0.02 g, hydroxypropyl-β-cyclodextrin 0.2 g, Brij-35 0.05 mL, guanidine hydrochloride 1 g, 3-sulfopropyltetradecyl dimethyl betaine 0.01 g, mannitol 1 g, ProClin 300 0.05 mL, with the remainder being water. Adjust the pH to 7.2–7.4.

[0041] The preparation method is the same as in Example 1.

[0042] Example 3 The dissociation agent, per 100 mL, consists of the following components: DTPA 0.2 g, salicylic acid 0.1 g, hydroxypropyl-β-cyclodextrin 1 g, Brij-35 0.2 mL, guanidine hydrochloride 2 g, 3-sulfopropyltetradecyl dimethyl betaine 0.1 g, mannitol 1 g, ProClin 300 0.05 mL, with the remainder being water. Adjust the pH to 7.2–7.4.

[0043] The preparation method is the same as in Example 1.

[0044] Comparative Example 1 The only difference from Example 1 is that hydroxypropyl-β-cyclodextrin has been removed.

[0045] Comparative Example 2 The only difference from Example 1 is that salicylic acid has been removed.

[0046] Comparative Example 3 The only difference from Example 1 is that mannitol and ProClin 300 have been removed.

[0047] Effect test The dissociation agents obtained in Examples 1-3 and Comparative Examples 1-3 were combined with streptavidin magnetic beads, biotinylated T3 or T4 analogs, and AP enzyme-labeled antibodies, respectively, to form detection reagents for Examples 1-3 and Comparative Examples 1-3. The following performance tests were performed: 1. Dissociation effect and sample comparison Thirty clinical serum samples each of TT3 and TT4, which had been measured using imported chemiluminescence reagent kits (Beckman), were collected. The Beckman TT3 and TT4 chemiluminescence detection systems were used with reagents 1-3 from the examples and 1-3 from the comparative examples, respectively. The detection results were compared with the values ​​measured using the imported reagents, and the relative deviation and correlation R² were calculated.

[0048] The results of TT3 detection in 30 serum samples (ng / mL) are compared as shown in Tables 1 and 2.

[0049] The comparison of TT4 test results (ug / dL) in 30 serum samples is shown in Tables 3 and 4.

[0050] Table 1

[0051] Table 2

[0052] Table 3

[0053] Table 4

[0054] The results in the table above show that: Using imported reagent kits as a reference, the samples tested by reagents 1-3 in this embodiment of the invention showed a correlation coefficient of R²=0.999 for TT3 and R²=0.999 for TT4. The relative deviation of all samples was controlled at an extremely low level (absolute value of TT3 ≤ 4.80%, absolute value of TT4 ≤ 4.83%), demonstrating extremely high consistency with imported reagents.

[0055] In contrast, Comparative Example 1, which lacked hydroxypropyl-β-cyclodextrin, and Comparative Example 2, which lacked salicylic acid, showed a significant decrease in the correlation between the detected values ​​of TT3 and TT4 and the imported reagents, and the relative deviation of multiple samples exceeded 30%, indicating that their dissociation was incomplete.

[0056] Comparative Example 3, which lacks stabilizers and preservatives, has detection performance comparable to Example 1, indicating that stabilizers and preservatives have no direct impact on the immediate dissociation effect of the sample.

[0057] 2. Precision Experiment Three levels of samples (low, medium, and high) were selected and measured 10 times (n=10) using the test reagent from Example 1. The mean, standard deviation (SD), and coefficient of variation (CV) were calculated. The results are shown in Table 5 - Precision test results.

[0058] Table 5

[0059] The results show that the dissociation agent of the present invention has good precision, and the CV of TT3 and TT4 detection is less than 3.0%, which meets the performance requirements of in vitro diagnostic reagents.

[0060] 3. Stability test (1) Long-term stability The reagents of Example 1 and Comparative Example 3 were stored at 2–8°C, and samples were taken at 0, 3, 6, 9, and 12 months. Low, medium, and high value samples were tested, and each sample was measured three times (n=3). At the same time, the appearance was observed and the pH was measured. The results are shown in Table 6—long-term stability test results.

[0061] Table 6

[0062] Relative deviation / % = (Measured value in the current month - Measured value in October) / Measured value in October × 100% (2) Accelerated stability test The reagents of Example 1 and Comparative Example 3 were placed at 37°C for 7 days for accelerated testing. Samples were taken on day 0 and day 7 for testing. Each sample was tested three times (n=3). The relative deviation was calculated. Microbial limits and appearance were also examined. The results are shown in Table 7 - Accelerated stability test results (37°C, 7 days).

[0063] Table 7

[0064] Relative deviation / % = (7-day measured value - 0-day measured value) / 0-day measured value × 100% Experimental results show that: Example 1, after being stored at 2–8°C for 12 months and accelerated at 37°C for 7 days, showed clear appearance, stable pH, and deviations of less than ±4.1% from the initial value, indicating stable performance. In contrast, Comparative Example 3 showed slight turbidity after 6 months of storage, increased detection deviation, and stratification after 12 months, indicating a significant decline in performance.

[0065] This indicates that the present invention further enhances the long-term stability and resistance to thermal degradation of the deionizing agent system through the synergistic effect of the stabilizer (mannitol) and the preservative (ProClin 300).

[0066] In summary, the dissociation agent provided by this invention achieves efficient and stable dissociation of serum TT3 and TT4 through the synergistic effect of its components. It also exhibits excellent compatibility with chemiluminescence detection systems, strong reagent stability, and high clinical application value and industrialization prospects.

[0067] While the embodiments disclosed in this invention are as described above, the content is merely for the purpose of facilitating understanding of the invention and is not intended to limit the invention. Any person skilled in the art to which this invention pertains may make any modifications and changes to the form and details of the implementation without departing from the spirit and scope disclosed herein; however, the scope of patent protection of this invention shall still be determined by the scope defined in the appended claims.

Claims

1. A dissociation agent, characterized in that, The dissociation agent comprises a chelating agent, a competitive binding agent, a clathrate agent, a denaturant, a surfactant system, a buffer system and a stabilizer. The chelating agent is DTPA; the competitive binding agent is salicylic acid; the clathrate agent is hydroxypropyl-β-cyclodextrin; and the denaturant is guanidine hydrochloride.

2. The dissociation reagent of claim 1, wherein, The surfactant system is a complex system of non-ionic surfactant and zwitterionic surfactant.

3. The dissociation reagent of claim 2, wherein, The non-ionic surfactant is Brij-35, and the zwitterionic surfactant is 3-sulfopropyl tetradecyl dimethyl betaine.

4. The dissociation reagent of claim 1, wherein, The buffer system is 10 mM-50 mM HEPES buffer, and the stabilizer is mannitol. Optionally, the dissociation agent further comprises a preservative, and the preservative is ProClin 300.

5. The dissociation agent according to any one of claims 1 to 4, characterized in that, The pH of the dissociation agent is 7.0-7.

5.

6. The dissociation reagent of claim 1, wherein, The dissociation agent, per 100 mL, comprises the following components: DTPA 0.1 g-0.2 g, salicylic acid 0.01 g-0.1 g, hydroxypropyl-β-cyclodextrin 0.2 g-1.0 g, Brij-35 0.05 mL-0.2 mL, guanidine hydrochloride 1.0 g-2.0 g, 3-sulfopropyl tetradecyl dimethyl betaine 0.01 g-0.1 g, stabilizer 0.5 g-2.0 g, preservative 0.01 mL-0.1 mL, and the balance is water; and the preservative is ProClin 300.

7. The dissociation reagent of claim 6, wherein, The dissociation agent, per 100 mL, comprises the following components: DTPA 0.15 g, salicylic acid 0.05 g, hydroxypropyl-β-cyclodextrin 0.5 g, Brij-35 0.1 mL, guanidine hydrochloride 1.5 g, 3-sulfopropyl tetradecyl dimethyl betaine 0.05 g, stabilizer 1 g, preservative 0.05 mL, and the balance is water; the pH of the dissociation agent is 7.2-7.4; and the preservative is ProClin 300.

8. The method for preparing the dissociation agent according to any one of claims 1-7, characterized in that, The method comprises the following steps: 1) providing a buffer, adding salicylic acid, and adjusting the pH to 7.0-7.5; 2) sequentially adding hydroxypropyl-β-cyclodextrin, DTPA, guanidine hydrochloride, zwitterionic surfactant and stabilizer to the solution obtained in step 1), wherein each component is fully stirred and dissolved when added; 3) adding non-ionic surfactant to the solution obtained in step 2) and mixing well; 4) adding a preservative, filtering, and obtaining the product.

9. A kit characterized in that, The dissociation agent of any one of claims 1-7.

10. Use of a dissociation agent according to any one of claims 1 to 7, characterized in that, The dissociation agent is used for preparing a kit for detecting one or both of total triiodothyronine and total thyroxine in serum.

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