Dissociation agent and kit

Abstract

Disclosed in the present invention are a dissociation agent and a detection kit for thyroid function detection. The dissociation agent comprises a protease and a reducing agent, and can effectively dissociate the bound forms of T3 and T4 in serum from binding proteins to form a free state. The dissociation agent and kit of the present invention are used for fluorescent immunochromatography detection, so that signal interference can be effectively reduced, and the sensitivity and accuracy of the detection can be improved.

Description

Dissociation agents and kits Technical Field

[0001] The present invention belongs to the technical field of biological detection, and relates to a dissociation agent and a kit for detecting TT3 and / or TT4 in a sample. Background Art

[0002] Triiodothyronine (T3) and thyroxine (T4) are major products secreted by the thyroid gland and are essential components of the hypothalamic-anterior pituitary-thyroid regulatory system. They influence anabolism and are crucial components of clinical thyroid function testing. T3 and T4 can aid in the diagnosis of thyroid cancer. In human blood, over 99% of T3 and T4 are bound to their respective binding proteins, primarily thyroid-binding globulin (TBG), with smaller amounts of albumin and thyroxine-binding prealbumin (TBPA). Bound T3 and T4 are difficult to detect. Dissociation agents are designed to dissociate bound T3 and T4 from their binding proteins, releasing them into free molecules for easier detection using relevant reagents. The degree of dissociation is closely related to the accuracy of the test. Therefore, when testing for total T3 (TT3) and total T4 (TT4) in serum, all bound T3 or T4 molecules must be completely dissociated to accurately determine the serum TT3 or TT4 content.

[0003] In recent years, rapid immunological diagnostic techniques and detection methods have been developed rapidly and widely used in a series of fields such as medical testing, drug analysis, environmental analysis, food analysis and biomedicine. With many advantages such as rapidity, simplicity, accuracy and stability, immunochromatography has become one of the commonly used techniques for clinical diagnosis today. Based on the principle of immunochromatography, the signal substance (hereinafter referred to as signal marker) used to label antibodies or antigens is accumulated on the detection line (T line) of the immunoassay test strip to generate a specific signal. The magnitude of the signal is related to the concentration of the analyte to be measured (antibody, antigen or hapten), thereby qualitatively and / or quantitatively detecting the analyte. Signal markers can be divided into colored colloidal particles (latex microspheres, colloidal gold, colloidal carbon, colloidal selenium, etc.), fluorescent microspheres, time-resolved fluorescent microspheres, colored fluorescent particles, quantum dots, upconversion luminescent materials (such as upconversion fluorescent materials and upconversion phosphorescent materials) and magnetic nanoparticles, etc. according to their types.

[0004] The primary dissociation agent used in TT3 / TT4 chemiluminescent reagent platforms from companies like Abbott and Beckman, and TT3 / TT4 enzyme-linked immunosorbent assay platforms from companies like Acon Biotech, is 8-phenylamino-1-naphthalenesulfonic acid ammonium salt (ANS). Fluorescence immunochromatographic platforms, as rapid diagnostic technologies capable of quantitatively detecting analytes, rely on their fluorescence signal to determine test accuracy. ANS solutions exhibit strong fluorescence, which can interfere with fluorescence immunochromatographic assays, leading to reduced sensitivity and inaccurate results.

[0005] Summary of the Invention

[0006] To address the shortcomings of the existing technology, the present invention provides a new dissociation agent formula that can effectively dissociate bound T3 and T4 in serum from their respective binding proteins. The free T3 or T4 content is then measured to obtain sample clinical experimental data, thereby improving the sensitivity and accuracy of clinical measurement of TT3 and TT4.

[0007] The present invention provides a dissociation agent for detecting TT3 and / or TT4 in a sample. The dissociation agent comprises a reducing agent and a protease for releasing bound T3 or T4 from a binding protein of T3 or T4.

[0008] The present invention also provides a kit for detecting TT3 and / or TT4 in a sample, wherein the kit comprises a dissociation agent, wherein the dissociation agent comprises a reducing agent and a protease for releasing bound T3 or T4 from the binding protein of T3 or T4.

[0009] In some embodiments of the present invention, the protease is selected from elastase or pepsin. In some embodiments of the present invention, the elastase is porcine pancreatic elastase. In some embodiments of the present invention, the concentration of the protease is 0.05-5 g / L, and the concentration of the reducing agent is 0.03-5 g / L. More specifically, the concentration of the protease is 0.1-2 g / L, and the concentration of the reducing agent is 0.1-3 g / L.

[0010] In some embodiments of the present invention, the reducing agent is a disulfide bond reducing agent, which is commonly used to reduce disulfide bonds in proteins and dissociates T3 and T4 from their binding state by disrupting the binding between T3 and T4 and their respective binding proteins. Specifically, the disulfide bond reducing agent can be selected from dithiothreitol (DTT), tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), or a combination thereof.

[0011] In some embodiments of the present invention, the dissociation agent further comprises a surfactant. In some embodiments of the present invention, the concentration of the surfactant is 0.01% to 5% (v / v), specifically, the concentration of the surfactant is 0.5% to 2% (v / v). In some embodiments of the present invention, the surfactant is a nonionic surfactant, such as Tween-20, Tween-21, Tween-40, Tween-60, Tween-61, Tween-80, Tween-81, Tween-85, Triton X-100, or a combination thereof.

[0012] In some embodiments of the present invention, the dissociation agent further comprises a buffer. In some embodiments of the present invention, the pH of the buffer is 6.5 to 8.5, specifically, the pH of the buffer is 7.0 to 8.5. In some embodiments of the present invention, the buffer is selected from PBS and Tris-HCl.

[0013] In some embodiments of the present invention, the dissociation agent further comprises an N-heterocyclic compound. In some embodiments of the present invention, the concentration of the N-heterocyclic compound is 0.02 to 5 g / L, specifically 0.1 to 2 g / L. In some embodiments of the present invention, the N-heterocyclic compound is selected from 5-bromo-2-methylpyridine, 3-bromo-5-hydroxypyridine, or a combination thereof. The N-heterocyclic compound dissociates T3 and T4 by competing for binding to their respective binding proteins in biological fluids such as blood, while also preventing the dissociated T3 and T4 from rebinding to their respective binding proteins.

[0014] In some embodiments of the present invention, the dissociating agent further comprises a preservative, and the concentration of the preservative is 0.01% to 2%, specifically, the concentration of the preservative is 0.1% to 2%. In some embodiments of the present invention, the preservative is selected from Proclin-150, Proclin-200, Proclin-300, Proclin-5000, sodium azide, thimerosal, sodium benzoate, or a combination thereof.

[0015] In some embodiments of the present invention, the kit further comprises a conjugate of a thyroid hormone or its analogue and a carrier protein, and a thyroid hormone antibody labeled with a fluorescent marker, wherein the thyroid hormone is selected from T3, T4, or a combination thereof. In some embodiments of the present invention, the conjugate of a thyroid hormone or its analogue and a carrier protein is coated on the detection line of an immunochromatographic test strip, and the thyroid hormone antibody labeled with a fluorescent marker is located upstream of the detection line during detection and migrates toward the detection line after the sample is added.

[0016] In some embodiments of the present invention, the fluorescent marker is selected from a time-resolved fluorescent molecule, a time-resolved fluorescent microsphere, a fluorescent compound, or a colored fluorescent microsphere. In some embodiments of the present invention, the fluorescent marker is a time-resolved fluorescent microsphere encapsulated with a lanthanide element or a chelate thereof. Furthermore, the kit may include a test card and instructions.

[0017] The present invention also provides a method for preparing a dissociation agent, comprising elastase, a reducing agent, a surfactant, an N-heterocyclic compound, and a buffer. During preparation, the buffer is first prepared and the pH is adjusted to 7.1 to 7.4. The elastase, reducing agent, and N-heterocyclic compound are then added and stirred until thoroughly dissolved. The surfactant is then added and stirred until thoroughly dissolved. Furthermore, the present invention has discovered that accurate detection of TT3 and TT4 in a sample can be achieved when the pH is adjusted to 7.0 to 8.5.

[0018] Beneficial effects: (1) The present invention provides a new dissociation agent formula, which can more fully dissociate the bound T3 and T4 in clinical samples from their respective binding proteins; (2) The present invention finds that the dissociation agent containing ANS used in the prior art will interfere with the fluorescent signal generated by the fluorescent marker on the detection line when used in the fluorescence chromatography platform, and the background fluorescence noise is large, thereby causing abnormal sample detection fluorescence signal values ​​and a large deviation in the overall detection concentration. The new dissociation agent formula provided by the present invention will not interfere with the fluorescent signal generated by the fluorescent marker on the detection line, and the background fluorescence noise is relatively small, thereby improving the sensitivity and accuracy of the fluorescence immunochromatography platform for detecting TT3 and TT4, and has a better clinical correlation with the detection value of the Abbott chemiluminescence reagent. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG1 is a schematic diagram of an exploded view of a test card used in the present invention.

[0020] FIG2 is a schematic diagram of an immunoassay strip in a test card used in the present invention. DETAILED DESCRIPTION

[0021] As shown in Figures 1 and 2, the test card includes a card cover 1, an immunoassay test strip 2, and a card holder 3. The immunoassay test strip 2 used is an immunochromatographic test strip, which includes a sample loading pad 21, a marking pad 22, a detection pad 25, and a sample absorption pad 26 that are overlapped together in sequence. The detection pad 25 is made of materials such as nitrocellulose, glass fiber, polyethersulfone, or nylon. For example, the detection pad 25 is a nitrocellulose membrane. The detection pad 25 is provided with a test line 23 and a control line 24. The sample loading pad 21 is made of a water-absorbing material, such as glass fiber or non-woven fabric. The marking pad 22 is also made of a water-absorbing material, such as polyester film, glass fiber, or non-woven fabric.

[0022] The immunoassay test strip 2 also includes a bottom support layer 27, which is made of a common hydrophobic material such as polyvinyl chloride to prevent the sample from leaking out of the bottom support layer 27. A detection pad 25 is disposed on the bottom support layer 27. A sample loading pad 21 is disposed on the bottom support layer 27, with one end of the sample loading pad 21 partially overlapping with the marking pad 22. A marking pad 22 is disposed on the bottom support layer 27, with one end of the marking pad 22 partially overlapping with the sample loading pad 21 and the other end of the marking pad 22 partially overlapping with the detection pad 25. A sample absorption pad 26 is disposed on the bottom support layer 27 and is made of a hydrophilic material, such as filter paper. One end of the sample absorption pad 26 partially overlaps with the detection pad 25. Furthermore, in some cases, the overlapping area between any two adjacent pads is 0.5 to 5 mm.

[0023] The immunoassay test strip 2 is located in a housing, which is composed of a card cover 1 and a card base 3 assembled by ultrasonic welding, snap fastening or gluing. In some cases, the card cover 1 and the card base 3 are made of plastic. A test paper slot 32 is provided in the middle of the card base 3 for inserting the immunoassay test strip 2. In some cases, the card cover 1 is provided with a plurality of snap fasteners extending downward (not shown in the figure), and the card base 3 is provided with a plurality of slots 31 extending upward. The snap fasteners provided on the card cover 1 and the slots 31 provided on the card base 3 correspond one to one, so that when the card cover 1, the immunoassay test strip 2 and the card base 3 are assembled together, the card cover 1 and the card base 3 can be firmly fixed together and the immunoassay test strip 2 can be fixed in the test paper slot 32. In some cases, the multiple snap fasteners on the card cover 1 are symmetrically distributed on both sides of the card cover 1, and the multiple slots 31 on the card base 3 are symmetrically distributed on both sides of the card base 3.

[0024] The card cover 1 is also provided with a sample addition port 11 and an observation window 12. When a clinical sample is added to the sample addition port 11, the sample enters the sample addition pad 21 located below the sample addition port 11. Under capillary action, the sample migrates along the length of the immunoassay test strip 2 toward the sample absorption pad 26. The observation window 12 is provided above the test line 23 and the control line 24 of the test pad 25. The excitation light emitted by the external light source 4 can be irradiated onto the test line 23 and the control line 24 of the immunoassay test strip 2 through the transparent or translucent observation window 12. After being irradiated by the excitation light, the emission light emitted by the fluorescent markers on the test line 23 and the control line 24 can also be output to the detector 7 through the observation window 12.

[0025] During testing, the test card is inserted into the test card insertion port of the analyzer. After the clinical sample is added to the test card, it is allowed to react outside the analyzer for a period of time before being inserted into the analyzer for testing. Alternatively, the test card can be immediately inserted into the analyzer after the clinical sample is added, and then allowed to react in the analyzer for a period of time before testing. The analyzer used in the present invention can be a commercially available analyzer, such as the dry-type fluorescent immunoassay analyzer FIC-Q100N (Suzhou Hemai Precision Instrument Co., Ltd.).

[0026] Depending on the analyte to be tested (such as an antigen, antibody, or hapten) and the immunoassay principle, the materials coated on the label pad 22 and the test line may vary. For example, the analyte to be tested is TT3, and the detection principle is a competitive assay. The label pad 22 is coated with fluorescently labeled T3 antibodies and fluorescently labeled rabbit IgG antibodies, the test line 23 is coated with a conjugate of the T3 antigen and the carrier protein BSA, and the control line 24 is coated with goat anti-rabbit IgG antibodies. When a clinical sample that has been previously treated with a dissociating agent is added to the sample pad 21 through the sample inlet 11, the clinical sample, carrying the fluorescently labeled T3 antibodies and fluorescently labeled rabbit IgG antibodies, flows along the length of the immunoassay test strip 2. When the clinical sample reaches label pad 22, the fluorescently labeled T3 antibody specifically binds to T3 in the clinical sample (if present). The resulting fluorescently labeled-T3 antibody-T3 complex continues to flow. The T3-BSA conjugate coated on test line 23 competes with the T3 in the clinical sample for the fluorescently labeled T3 antibody. Illuminating test line 23 with excitation light from the analyzer's light source generates a test signal. When the sample flows to control line 24, the fluorescently labeled rabbit IgG antibody specifically binds to the goat anti-rabbit IgG antibody coated on control line 24, forming a complex that is immobilized on control line 24. Illuminating control line 24 with excitation light from the analyzer's light source generates a control signal.

[0027] In view of the fact that the fluorescent signal generated by the control line in the present invention can only serve to indicate whether the added clinical sample flows to the sample absorption pad 26 and does not participate in the calculation of the analyte concentration, the fluorescent marker in the fluorescent marker-labeled rabbit IgG antibody can also be replaced by colored colloidal particles such as latex microspheres, colloidal gold, colloidal carbon, colloidal selenium, etc.

[0028] The fluorescent marker used in the present invention can be a time-resolved fluorescent marker. Time-resolved fluorescent markers have the characteristic of delayed luminescence, which means that when the excitation light emitted by the external light source is turned off, they can still continue to emit fluorescence for a certain period of time. The time-resolved fluorescent marker can exist in molecular form, called time-resolved fluorescent molecules, which can be selected from lanthanides such as samarium (Sm(III)), dysprosium (Dy(III)), europium (Eu(III))) and terbium (Tb(III)) and their chelates. A suitable lanthanide chelate is N-(p-isothiocyanatophenyl)-diethylenetriaminetetraacetic acid-Eu +3 . Time-resolved fluorescent markers can also exist in another form: time-resolved fluorescent microspheres, that is, time-resolved fluorescent molecules are encapsulated in the interior or surface of natural or synthetic microspheres or microbeads. Each time-resolved fluorescent microsphere can encapsulate thousands of fluorescent molecules, effectively improving the detection sensitivity. The fluorescent marker in the present invention can be selected from time-resolved fluorescent microspheres with a diameter of 100nm to 400nm, which encapsulate europium chelates inside and carry carboxyl groups on the surface (Suzhou Weidu Biotechnology Co., Ltd., product number FT0200CA), which have an excitation wavelength of 360nm and an emission wavelength of 615nm.

[0029] The fluorescent marker used in the present invention can also be a fluorescent compound that can produce a fluorescent signal without luminescence delay characteristics when irradiated with excitation light of a suitable wavelength, and can be selected from quantum dots; fluorescein and its derivatives, such as fluorescein isothiocyanate (FITC); fluorescent proteins and improved variants thereof, such as green fluorescent protein, red fluorescent protein, blue fluorescent protein, yellow fluorescent protein, orange fluorescent protein, etc.

[0030] The fluorescent marker used in the present invention may also be a colored fluorescent microsphere, i.e., a microsphere or microbead coated with a fluorescent compound without a luminescence delay characteristic, which produces a fluorescent signal without a luminescence delay characteristic when irradiated with excitation light of a suitable wavelength. The colored fluorescent microsphere can be selected from green fluorescent microspheres, blue fluorescent microspheres, red fluorescent microspheres, yellow fluorescent microspheres, and colored fluorescent microspheres (emitting fluorescence of various specific colors).

[0031] Whether in time-resolved fluorescent microspheres or colored fluorescent microspheres, the polymer forming the microspheres or microbeads can be selected from polystyrene, butadiene styrene, styrene acrylic acid-ethylene terpolymer, polymethyl methacrylate, polyethyl methyl acrylate, styrene-maleic anhydride copolymer, polyvinyl acetate, polyvinyl pyridine, polydivinylbenzene, polybutylene terephthalate, acrylonitrile, vinyl chloride-acrylate, etc., or their aldehyde, carboxyl, amino, hydroxyl, hydrazide derivatives, or mixtures thereof. In addition, the surface of the microspheres or microbeads usually carries hydroxyl, carboxyl, amino, aldehyde, sulfonic groups, etc., which can be coupled to antibodies or antigens or hapten-carrier protein conjugates through conventional chemical coupling reagents. In some cases, the particle size of the time-resolved fluorescent microspheres is 20 nm to 100 μm; the particle size of the colored fluorescent microspheres is 100 nm to 100 μm.

[0032] The following examples further illustrate the present invention. These examples are not intended to limit the scope of the present invention, but to provide a further understanding of the present invention.

[0033] Example 1: Dissociation agent formulation

[0034] Dissociation agent formula: elastase 0.1-2 g / L; reducing agent 0.1-3 g / L; N-heterocyclic compound 0.1-2 g / L; surfactant 0.5%-2% (v / v); buffer solution with a pH of 7.1-7.4.

[0035] Formulation 1: 0.1 g elastase, 1.0 g DTT, 2.0 g 5-bromo-2-methylpyridine, 0.5% (v / v) Tween-40, and 10 mM PBS buffer (pH 7.1-7.4) in 1 L deionized water.

[0036] Formulation 2: 1.0 g elastase, 3.0 g DTT, 0.1 g 5-bromo-2-methylpyridine, 0.5% (v / v) Tween-40, and 10 mM PBS buffer (pH 7.1-7.4) in 1 L deionized water.

[0037] Formulation 3: 2.0 g elastase, 0.1 g DTT, 1.0 g 3-bromo-5-hydroxypyridine, Tween-40 2% (v / v), and 10 mM PBS buffer (pH 7.1-7.4) in 1 L deionized water.

[0038] In addition, DTT in the formula can also be replaced by TCEP-HCl; the surfactant in the formula can also be selected from Tween-20, Tween-21, Tween-60, Tween-61, Tween-80, Tween-81, Tween-85 and Triton X-100; the buffer salt in the formula can also be selected from 50mM Tris-HCl buffer (pH 7.1-7.4).

[0039] In order to maintain the stability of the dissociator formulation during storage, a preservative may be added to the dissociator formulation. The preservative may be selected from sodium azide, thimerosal, sodium benzoate, Proclin-150, Proclin-200, Proclin-300 and Proclin-5000, and its concentration may be selected to be 0.1% to 2% (v / v).

[0040] Example 2: Preparation method of dissociating agent

[0041] A method for preparing a dissociation agent for detecting TT3 and TT4 in human serum comprises the following steps:

[0042] First, prepare the buffer solution in Example 1 and adjust the pH to 7.1-7.4. Then, add the elastase, reducing agent, N-heterocyclic compound and preservative in Example 1 and stir to mix. After complete mixing and dissolution, add the surfactant and stir to mix.

[0043] Example 3: TT3 performance test

[0044] Thirty clinical serum samples (including low, medium, and high values) were collected using the Abbott Total Triiodothyronine Assay Kit (chemiluminescent microparticle immunoassay) (National Medical Device Registration No. 20142405914) and the Abbott ARCHITECT i2000 immunoassay analyzer. Samples were numbered 1 to 30. For each sample, 40 μL was pipetted into five reaction tubes, numbered A, B, C, D, and E plus the sample number: A1-A30, B1-B30, C1-C30, D1-D30, and E1-E30.

[0045] Add 100 μL of the dissociation agent formula 1 in Example 1 to each reaction tube in group A;

[0046] Add 100 μL of dissociation agent formula 2 in Example 1 to each reaction tube in group B;

[0047] Add 100 μL of dissociation agent formula 3 in Example 1 to each reaction tube in group C;

[0048] Add 100 μL of the control dissociation agent formula to each reaction tube in Group D. The specific formula is as follows:

[0049] In 1 L of deionized water, 0.5 g EDTA, 2.5 g 5-bromo-2-methylpyridine, 2.5 g 3-bromo-5-hydroxypyridine, 1 ml Proclin-300, 5 ml Tween-40, 10 g ANS, and 1 g sodium citrate were added.

[0050] Add 100 μL of conventional treatment solution to each reaction tube in Group E. The specific formula is as follows:

[0051] Add 1 ml of Proclin-300, 5 ml of Tween-40, 2.9 g of disodium hydrogen phosphate, and 0.296 g of sodium dihydrogen phosphate to 1 L of deionized water.

[0052] After mixing the above reaction tube solutions, the mixture was allowed to stand at room temperature for 10 minutes. The total triiodothyronine (T3) quantitative detection kit (fluorescent immunoassay) reagent (item number F131-20211) produced by Aikon Biotechnology (Hangzhou) Co., Ltd. was used in conjunction with a dry fluorescent immunoassay analyzer (FIC-Q100N) produced by Suzhou Hemai Precision Instrument Co., Ltd. The test results were compared with the fixed value results of the total triiodothyronine determination kit (chemiluminescent microparticle immunoassay) produced by Abbott (National Medical Device Injection No. 20142405914) in conjunction with Abbott's immunoassay analyzer ARCHITECT i2000 (hereinafter referred to as Abbott's chemiluminescent detection value). The results are shown in Table 1.

[0053] Table 1 TT3 comparison test results (unit: nmol / L)

[0054] From the analysis of the comparison results in Table 1, the conclusions are as follows:

[0055] 1. After treating clinical serum samples with conventional treatment solution (Group E), the measured values ​​were generally much lower. This is because most of the TT3 in the clinical samples was not dissociated and thus could not be bound by the paired antibody. For example, the detection value of sample No. 30 with Abbott chemiluminescence reagent was 9.84nmol / L, the detection value of Group A was 9.11nmol / L, the detection value of Group B was 9.45nmol / L, the detection value of Group C was 9.26nmol / L, and the detection value of Group E was only 1.35nmol / L.

[0056] 2. After treating clinical samples with the treatment solution containing the dissociation agent ANS (Group D), the test values ​​of clinical samples were generally higher than those treated with the conventional treatment solution (Group E). However, because ANS itself interferes with the fluorescence signal value on the fluorescence chromatography platform, the background fluorescence noise is high, resulting in abnormal fluorescence signal values ​​in clinical samples and a significant deviation from the overall detection concentration. In terms of clinical correlation with the detection values ​​of Abbott's chemiluminescence reagent, the correlations of Groups AC were 0.9873, 0.9880, and 0.9825, respectively, while the correlation of Group D was only 0.7304.

[0057] 3. The correlation between the values ​​detected by the dissociation reagent in group AC and those detected by Abbott's chemiluminescence reagent was good, both greater than 0.95. This is because the dissociation reagent prepared in group AC fully dissociates TT3 in clinical samples and does not affect the detection of fluorescence signal values.

[0058] Example 4: TT4 performance test

[0059] Thirty clinical serum samples (including low, medium, and high values) were collected using the Abbott Total Thyroxine Assay Kit (chemiluminescent microparticle immunoassay) (National Medical Device Registration No. 20162404093) and the ARCHITECT i2000 instrument. Samples were numbered 1 to 30. For each sample, 40 μL was pipetted into five reaction tubes, numbered A, B, C, D, and E. The tubes were then numbered A1 to A30, B1 to B30, C1 to C30, D1 to D30, and E1 to E30, respectively.

[0060] Add 100 μL of the dissociation agent formula 1 in Example 1 to each reaction tube in group A;

[0061] Add 100 μL of dissociation agent formula 2 in Example 1 to each reaction tube in group B;

[0062] Add 100 μL of dissociation agent formula 3 in Example 1 to each reaction tube in group C;

[0063] Add 100 μL of the control dissociation agent formula to each reaction tube in Group D. The specific formula is as follows:

[0064] In 1 L of deionized water, 0.5 g EDTA, 2.5 g 5-bromo-2-methylpyridine, 2.5 g 3-bromo-5-hydroxypyridine, 1 ml Proclin-300, 5 ml Tween-40, 10 g ANS, and 1 g sodium citrate were added.

[0065] Add 100 μL of conventional treatment solution to each reaction tube in Group E. The specific formula is as follows:

[0066] Add 1 ml of Proclin-300, 5 ml of Tween-40, 2.9 g of disodium hydrogen phosphate, and 0.296 g of sodium dihydrogen phosphate to 1 L of deionized water.

[0067] After mixing the above reaction tube solutions, the solutions were allowed to stand at room temperature for 10 minutes, and then the total thyroxine (T4) quantitative detection kit (fluorescence immunoassay) reagent registered by Aikon Biotechnology (Hangzhou) Co., Ltd. (Cat. No. F131-202311) was used in conjunction with a dry-type fluorescence immunoassay analyzer (FIC-Q100N) produced by Suzhou Hemai Precision Instrument Co., Ltd. The test results were compared with the fixed value results of the total thyroxine determination kit (chemiluminescent microparticle immunoassay) produced by Abbott (National Medical Device Injection No. 20162404093) in conjunction with Abbott's immunoassay analyzer ARCHITECT i2000 (hereinafter referred to as Abbott's chemiluminescence detection value). The results are shown in Table 2.

[0068] Table 2 TT4 comparison test results (unit: nmol / L)

[0069] From the analysis of the comparison results in Table 2, the conclusions are as follows:

[0070] 1. After treating clinical serum samples with conventional treatment solution (Group E), the measured values ​​were generally significantly lower. This is because the majority of TT4 in the clinical sample was not dissociated and thus unavailable for binding by the paired antibody. For example, the Abbott chemiluminescence reagent detected the concentration of sample No. 26 at 196.82 nmol / L, while the values ​​for Group A were 215.00 nmol / L, Group B were 195.65 nmol / L, Group C were 231.70 nmol / L, and Group E was only 1.72 nmol / L.

[0071] 2. After treating samples with the treatment solution containing the dissociating agent ANS (Group D), the measured values ​​of clinical samples were generally higher than those treated with the conventional treatment solution (Group E). However, because ANS itself interferes with the fluorescence signal value on the fluorescence chromatography platform, the background fluorescence noise is high, resulting in abnormal fluorescence signal values ​​and a significant deviation from the overall detection concentration. In terms of clinical correlation with the detection values ​​of Abbott's chemiluminescence reagent, the correlations for Groups AC were 0.9819, 0.9797, and 0.9778, respectively, while the correlation for Group D was only 0.7796.

[0072] 2. After treating clinical samples with the treatment solution containing the dissociation agent ANS (Group D), the test values ​​of clinical samples were generally higher than those treated with the conventional treatment solution (Group E). However, because ANS itself interferes with the fluorescence signal value on the fluorescence chromatography platform, the background fluorescence noise is high, resulting in abnormal fluorescence signal values ​​in clinical samples and a significant deviation from the overall detection concentration. In terms of clinical correlation with the detection values ​​of Abbott's chemiluminescence reagent, the correlations of Groups AC were 0.9873, 0.9880, and 0.9825, respectively, while the correlation of Group D was only 0.7304.

[0073] 3. The correlation between the values ​​detected by the dissociation reagent in group AC and those detected by Abbott's chemiluminescence reagent was good, both greater than 0.95. This is because the dissociation reagent prepared in group AC fully dissociates TT4 in clinical samples and does not affect the detection of fluorescence signal values.

[0074] Example 5: Reducing agent evaluation experiment

[0075] Formulation 4: 0.1 g elastase, 1.0 g TCEP-HCl, 2.0 g 5-bromo-2-methylpyridine, 0.5% (v / v) Tween-40, and 10 mM PBS buffer (pH 7.1-7.4) in 1 L deionized water.

[0076] Formulation 5: 0.1 g elastase, 3.0 g TCEP-HCl, 2.0 g 5-bromo-2-methylpyridine, 0.5% (v / v) Tween-40, and 10 mM PBS buffer (pH 7.1-7.4) in 1 L deionized water.

[0077] Fifteen fresh clinical blood samples were collected using the Abbott Total Triiodothyronine Assay Kit (chemiluminescent microparticle immunoassay) (National Medical Device Registration No. 20142405914) and the Abbott ARCHITECT i2000 immunoassay analyzer. The samples were then tested using the TT3 performance test method described in Example 3, except that only three reaction tubes were selected. 100 μL of Dissociation Agent Formula 1 from Example 1 was added to reaction tube A, 100 μL of Dissociation Agent Formula 4 was added to reaction tube B, and 100 μL of Dissociation Agent Formula 5 was added to reaction tube B. The results are shown in Table 3.

[0078] Table 3 Reducing agent evaluation test results (unit: nmol / L)

[0079] As shown in Table 3, after selecting dissociation agent formula 1, formula 4 and formula 5, the correlation coefficients between the concentration values ​​calculated by fluorescence immunoassay and the Abbott chemiluminescence detection values ​​were all greater than 0.95, indicating that dissociation agent formula 1, formula 4 and formula 5 can fully dissociate TT3 in the sample, thereby making the detected TT3 values ​​in the sample more accurate.

[0080] Fifteen fresh clinical blood samples were collected using a total thyroxine assay kit (chemiluminescent microparticle immunoassay) manufactured by Abbott Laboratories (National Medical Device Registration No. 20162404093) and an instrument model (ARCHITECT i2000). The concentration distribution was required to be uniform. The TT4 performance test method described in Example 4 was then used for testing, except that only three reaction tubes were selected. 100 μL of dissociation agent formula 1 from Example 1 was added to the reaction tube in Group A, 100 μL of dissociation agent formula 4 was added to the reaction tube in Group B, and 100 μL of dissociation agent formula 5 was added to the reaction tube in Group B. The results are shown in Table 4.

[0081] Table 4 Reducing agent evaluation test results (unit: nmol / L)

[0082] As can be seen from Table 4, after selecting dissociation agent formula 1, formula 4 and formula 5, the correlation coefficients between the concentration values ​​calculated by fluorescence immunoassay and the Abbott chemiluminescence detection values ​​are all greater than 0.95, which indicates that dissociation agent formula 1, formula 4 and formula 5 can fully dissociate TT4 in the sample, thereby making the detected TT4 value in the sample more accurate.

[0083] Example 6: Surfactant evaluation experiment

[0084] Formulation 6: 0.1 g elastase, 1.0 g DTT, 2.0 g 5-bromo-2-methylpyridine, 0.5% (v / v) Tween-20, and 10 mM PBS buffer (pH 7.1-7.4) in 1 L deionized water.

[0085] Formulation 7: 0.1 g elastase, 1.0 g DTT, 2.0 g 5-bromo-2-methylpyridine, 0.5% (v / v) Triton X-100, and 10 mM PBS buffer (pH 7.1-7.4) in 1 L deionized water.

[0086] Fifteen fresh clinical blood samples were collected using the Abbott Total Triiodothyronine Assay Kit (chemiluminescent microparticle immunoassay) (National Medical Device Registration No. 20142405914) and the Abbott ARCHITECT i2000 immunoassay analyzer. The samples were then tested using three different TT3 performance testing methods, with the difference being that only three reaction tubes were used. In addition, 100 μL of Dissociation Agent Formula 1 from Example 1 was added to reaction tube A, 100 μL of Dissociation Agent Formula 6 was added to reaction tube B, and 100 μL of Dissociation Agent Formula 7 was added to reaction tube B. The results are shown in Table 5.

[0087] Table 5 Surfactant evaluation test results (unit: nmol / L)

[0088] As shown in Table 5, after selecting dissociation agent formula 1, formula 6 and formula 7, the correlation coefficients between the concentration values ​​calculated by fluorescence immunoassay and the Abbott chemiluminescence detection values ​​were all greater than 0.95, indicating that dissociation agent formula 1, formula 6 and formula 7 can fully dissociate TT3 in the sample, thereby making the detected TT3 values ​​in the sample more accurate.

[0089] Fifteen fresh clinical blood samples were collected using a total thyroxine assay kit (chemiluminescent microparticle immunoassay) manufactured by Abbott Laboratories (National Medical Device Registration No. 20162404093) and an instrument model (ARCHITECT i2000). The concentration distribution was required to be uniform. The TT4 performance test method described in Example 4 was then used for testing, except that only three reaction tubes were selected. 100 μL of dissociation agent formula 1 from Example 1 was added to the reaction tube in Group A, 100 μL of dissociation agent formula 6 was added to the reaction tube in Group B, and 100 μL of dissociation agent formula 7 was added to the reaction tube in Group B. The results are shown in Table 6.

[0090] Table 6 Surfactant evaluation test results (unit: nmol / L)

[0091] As can be seen from Table 6, after selecting dissociation agent formula 1, formula 4 and formula 5, the correlation coefficients between the concentration values ​​calculated by fluorescence immunoassay and the Abbott chemiluminescence detection values ​​are all greater than 0.95, which indicates that dissociation agent formula 1, formula 6 and formula 7 can fully dissociate TT4 in the sample, thereby making the detected TT4 value in the sample more accurate.

[0092] Example 7: Buffer evaluation experiment

[0093] Formulation 8: 0.1 g elastase, 1.0 g DTT, 2.0 g 5-bromo-2-methylpyridine, 1.0% (v / v) Tween-40, and 50 mM Tris-HCl buffer (pH 7.1-7.4) in 1 L deionized water.

[0094] Fifteen fresh clinical blood samples were collected using the Abbott Total Triiodothyronine Assay Kit (chemiluminescent microparticle immunoassay) (National Medical Device Registration No. 20142405914) and the Abbott ARCHITECT i2000 immunoassay analyzer. The samples were then tested using the TT3 performance test method described in Example 3, with the difference that only two reaction tubes were used. 100 μL of Dissociation Agent Formula 1 from Example 1 was added to reaction tube A, and 100 μL of Dissociation Agent Formula 8 was added to reaction tube B. The results are shown in Table 7.

[0095] Table 7 Buffer evaluation test results (unit: nmol / L)

[0096] As shown in Table 7, after selecting dissociation agent formula 1 and formula 8, the correlation coefficients between the concentration values ​​calculated by fluorescence immunoassay and the Abbott chemiluminescence detection values ​​were both greater than 0.95, indicating that dissociation agent formula 1 and formula 8 can fully dissociate TT3 in the sample, thereby making the detected TT3 values ​​in the sample more accurate.

[0097] Fifteen fresh clinical blood samples were collected using a total thyroxine assay kit (chemiluminescent microparticle immunoassay) manufactured by Abbott (National Medical Device Registration No. 20162404093) and an instrument model (ARCHITECT i2000). The concentration distribution was required to be uniform. The TT4 performance test method described in Example 4 was then used for testing, except that only three reaction tubes were selected. 100 μL of Dissociation Agent Formula 1 from Example 1 was added to the reaction tube in Group A, and 100 μL of Dissociation Agent Formula 8 was added to the reaction tube in Group B. The results are shown in Table 8.

[0098] Table 8 Buffer evaluation test results (unit: nmol / L)

[0099] As can be seen from Table 8, after selecting dissociation agent formula 1 and formula 8, the correlation coefficients between the concentration values ​​calculated by fluorescence immunoassay and the Abbott chemiluminescence detection values ​​were both greater than 0.95, indicating that dissociation agent formula 1 and formula 8 can fully dissociate TT4 in the sample, thereby making the detected TT4 values ​​in the sample more accurate.

[0100] Example 8: Dissociation Agent Evaluation Experiment

[0101] Recipe 9: 0.1 g elastase, 0.5% (v / v) Tween-40, 10 mM PBS (pH 7.1-7.4) in 1 L deionized water;

[0102] Formulation 10: 1.0 g elastase, 0.5% (v / v) Tween-40, 10 mM PBS buffer (pH 7.1-7.4) in 1 L deionized water;

[0103] Formulation 11: 0.1 g elastase, 0.1 g DTT, 0.5% (v / v) Tween-40, 10 mM PBS buffer (pH 7.1-7.4) in 1 L deionized water;

[0104] Formulation 12: 0.1 g elastase, 1.0 g DTT, 0.5% (v / v) Tween-40, 10 mM PBS buffer (pH 7.1-7.4) in 1 L deionized water;

[0105] Formulation 13: 1.0 g elastase, 0.1 g DTT, 0.5% (v / v) Tween-40, 10 mM PBS buffer (pH 7.1-7.4) in 1 L deionized water;

[0106] Formulation 14: 1.0 g elastase, 1.0 g DTT, 0.5% (v / v) Tween-40, 10 mM PBS buffer (pH 7.1-7.4) in 1 L deionized water.

[0107] Five fresh clinical blood samples were collected using the Abbott Total Triiodothyronine Assay Kit (chemiluminescent microparticle immunoassay) (National Medical Device Injection No. 20142405914) and the Abbott Immunoassay Analyzer ARCHITECT i2000, with a uniform concentration distribution. The TT3 performance test method in Example 3 was then used for testing, with the difference being that only six reaction tubes were selected, and 100 μL of dissociation agent formulas 9, 10, 11, 12, 13, and 14 were added to the reaction tubes in Groups A, B, C, D, E, and F, and 100 μL of the control dissociation agent formula in Example 3 was added to the reaction tube in Group G. The results are shown in Table 9.

[0108] Table 9 Dissociation agent evaluation test results (unit: nmol / L)

[0109] As shown in Table 9, in general, compared with the control dissociation agent formulation, dissociation agent formulations 11 to 14 can significantly improve the accuracy of the detected TT3 values.

[0110] Five fresh clinical blood samples were collected using a total thyroxine assay kit (chemiluminescent microparticle immunoassay) produced by Abbott Laboratories (National Medical Device Injection No. 20162404093) and an instrument model (ARCHITECT i2000), with a uniform concentration distribution. The test was then performed according to the TT4 performance test method in Example 3, except that only six reaction tubes were selected, and 100 μL of dissociation agent formulas 9, 10, 11, 12, 13, and 14 were added to the reaction tubes in groups A, B, C, D, E, and F, and 100 μL of the control dissociation agent formula in Example 3 was added to the reaction tube in group G. The results are shown in Table 10.

[0111] Table 10 Dissociation agent evaluation test results (unit: nmol / L)

[0112] As shown in Table 10, in general, compared with the control dissociation agent formulation, dissociation agent formulations 11 to 14 can significantly improve the accuracy of the detected TT4 values.

Claims

1. A dissociating agent, characterized in that The dissociation agent includes a reducing agent and a protease that releases bound T3 or T4 from the binding protein of T3 or T4.

2. The dissociating agent according to claim 1, characterized in that The concentration of the protease is 0.05-5 g / L, preferably 0.1-2 g / L, preferably, the protease is selected from elastase or pepsin; the concentration of the reducing agent is 0.03-5 g / L, preferably 0.1-3 g / L, preferably, the reducing agent is a disulfide bond reducing agent, preferably, the disulfide bond reducing agent is selected from dithiothreitol, tris(2-carboxyethyl)phosphine hydrochloride or a combination thereof.

3. The dissociating agent according to claim 1, characterized in that The dissociation agent also includes a surfactant. Preferably, the concentration of the surfactant is 0.01% to 5% (v / v), preferably 0.5% to 2% (v / v). Preferably, the surfactant is a non-ionic surfactant. Preferably, the non-ionic surfactant is selected from Tween-20, Tween-21, Tween-40, Tween-60, Tween-61, Tween-80, Tween-81, Tween-85, Triton X-100 or a combination thereof.

4. The dissociating agent according to claim 1, characterized in that The dissociation agent further comprises a buffer solution, wherein the pH value of the buffer solution is 6.5 to 8.5, preferably, the pH value of the buffer solution is 7.0 to 8.5, and preferably, the buffer solution is selected from PBS and Tris-HCl.

5. The dissociating agent according to claim 1, characterized in that The dissociator also includes an N-heterocyclic compound. Preferably, the concentration of the N-heterocyclic compound is 0.02 to 5 g / L, preferably 0.1 to 2 g / L. Preferably, the N-heterocyclic compound is selected from 5-bromo-2-methylpyridine, 3-bromo-5-hydroxypyridine or a combination thereof.

6. The dissociating agent according to claim 1, characterized in that The dissociating agent also includes a preservative, the concentration of the preservative is 0.01-2%, preferably 0.1%-2%. Preferably, the preservative is selected from Proclin-150, Proclin-200, Proclin-300, Proclin-5000, sodium azide, thimerosal, sodium benzoate or a combination thereof.

7. A kit, characterized in that: The kit comprises the dissociation agent according to any one of claims 1 to 6. Preferably, the kit further comprises a conjugate of a thyroid hormone or its analogue with a carrier protein and a thyroid hormone antibody labeled with a fluorescent marker, wherein the thyroid hormone is selected from T3, T4 or a combination thereof.

8. The kit according to claim 7, characterized in that The conjugate of thyroid hormone or its analogue and carrier protein is coated on the detection line of the immunochromatographic test strip. The thyroid hormone antibody labeled with a fluorescent marker is located upstream of the detection line during detection and moves toward the detection line after the sample is added.

9. The kit according to claim 7, characterized in that The fluorescent marker is selected from time-resolved fluorescent molecules, time-resolved fluorescent microspheres, fluorescent compounds or colored fluorescent microspheres.

10. The kit according to claim 7, characterized in that The fluorescent marker is a time-resolved fluorescent microsphere encapsulated with europium chelate.

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