Dissociation agent and kit

Abstract

The invention discloses a dissociation agent and a detection kit for thyroid function detection, the dissociation agent comprises protease and a reducing agent, and the dissociation agent can well dissociate T3 and T4 in a binding state from binding protein into a free state. The dissociation agent and the kit are applied to fluorescence immunochromatography detection, signal interference can be effectively reduced, and the detection sensitivity and accuracy are improved.

Description

Technical Field

[0001] The 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 the main products of thyroid secretion, and are also indispensable components of the integrity of the hypothalamus-anterior pituitary-thyroid regulation system. They have an impact on anabolism and are important components of clinical thyroid function testing. T3 and T4 can assist in the diagnosis of thyroid cancer. In human blood, more than 99% of T3 and T4 are bound to their respective binding proteins, mainly to thyroid binding globulin (TBG) and a small amount of albumin and thyroxine-binding prealbumin (TBPA). Bound T3 and T4 are difficult to detect. The main function of the dissociator is to dissociate the bound T3 and T4 from their respective binding proteins to form free molecules, so that the relevant reagents can detect them. The degree of dissociation is closely related to the accuracy of the actual test. Therefore, when detecting total T3 (TT3) and total T4 (TT4) in serum, all bound T3 or T4 molecules must be dissociated so that the accurate content of TT3 or TT4 in serum can be obtained.

[0003] In recent years, rapid immunological diagnostic techniques and detection methods have been rapidly developed 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 gathered on the detection line (T line) of the immunoassay test strip to generate a specific signal, and the size of the signal is related to the concentration of the analyte (antibody, antigen or hapten) to be measured, 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 different types.

[0004] The main component of the dissociation agent currently used in TT3 / TT4 chemiluminescent reagent platforms such as Abbott and Beckman, and TT3 / TT4 enzyme-linked immunosorbent platforms such as Acon Biotechnology is 8-phenylamino-1-naphthalenesulfonic acid ammonium salt (ANS). Fluorescence immunochromatography platform, as a rapid diagnostic technology that can quantitatively detect the substance being tested, its fluorescence signal value determines the accuracy of the test. ANS solution itself has strong fluorescence, which has a certain background interference with the detection of fluorescence immunochromatography platform, resulting in reduced sensitivity and inaccurate results. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a new dissociation agent formula, which can effectively dissociate the bound T3 and T4 in serum from their respective binding proteins, and then obtain sample clinical experimental data by measuring the content of free T3 or T4, so as to improve the sensitivity and accuracy of clinical measurement of TT3 and TT4.

[0006] 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.

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

[0008] 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 elastase from porcine pancreas. 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.

[0009] In some embodiments of the present invention, the reducing agent is a disulfide bond reducing agent, which is commonly used for reducing disulfide bonds in proteins, and dissociates T3 and T4 from the binding state by destroying 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.

[0010] 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.

[0011] 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.

[0012] 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, the concentration of the N-heterocyclic compound is 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, and at the same time, it can also prevent T3 and T4 in the dissociated state from binding to their respective binding proteins again.

[0013] In some embodiments of the present invention, the dissociation 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.

[0014] In some embodiments of the present invention, the kit further comprises a conjugate of 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 thyroid hormone or its analogue and a carrier protein is coated on the detection line of the immunochromatographic test strip, and 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.

[0015] In some embodiments of the present invention, the fluorescent marker is selected from time-resolved fluorescent molecules, time-resolved fluorescent microspheres, fluorescent compounds or colored fluorescent microspheres. In some embodiments of the present invention, the fluorescent marker is a time-resolved fluorescent microsphere encapsulated with a lanthanide element or its chelate. In addition, the kit may also include a test card and instructions, etc.

[0016] The present invention also provides a method for preparing a dissociating agent, wherein the dissociating agent comprises elastase, a reducing agent, a surfactant, an N-heterocyclic compound and a buffer. During the preparation, the buffer is first prepared and the pH is adjusted to 7.1 to 7.4, then the elastase, the reducing agent and the N-heterocyclic compound are added and stirred, and after the mixture is completely mixed and dissolved, the surfactant is added and stirred. In addition, the present invention also finds that when the pH is adjusted to 7.0 to 8.5, TT3 and TT4 in the sample can also be accurately detected.

[0017] 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 fluorescent chromatography platform, and the background fluorescence noise is large, thereby causing abnormal sample detection fluorescence signal values ​​and causing 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 fluorescent 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

[0018] Figure 1 . Schematic diagram of the decomposition of the test card used in the present invention.

[0019] Figure 2 . Schematic diagram of the immunoassay strip in the test card used in the present invention. DETAILED DESCRIPTION

[0020] like Figure 1 and Figure 2 As shown, 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 which are overlapped 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. A detection line 23 and a control line 24 are provided on the detection pad 25. The sample loading pad 21 is made of a water-absorbing material, and glass fiber or non-woven fabric can be selected. The marking pad 22 is also made of a water-absorbing material, and polyester film, glass fiber or non-woven fabric can be selected.

[0021] The immunoassay test strip 2 also includes a bottom support layer 27, which is made of common hydrophobic materials such as polyvinyl chloride to ensure that the sample cannot leak out of the bottom support layer 27. The detection pad 25 is arranged on the bottom support layer 27. The sample loading pad 21 is arranged on the bottom support layer 27, and one end of the sample loading pad 21 partially overlaps with the marking pad 22; the marking pad 22 is arranged on the bottom support layer 27, and one end of the marking pad 22 partially overlaps with the sample loading pad 21, and the other end of the marking pad 22 partially overlaps with the detection pad 25; the sample absorption pad 26 is arranged on the bottom support layer 27, and is made of a hydrophilic material, which can be selected from filter paper; one end of the sample absorption pad 26 partially overlaps with the detection pad 25. In addition, in some cases, the overlapping area between any two adjacent pads is 0.5 to 5 mm long.

[0022] The immunoassay test strip 2 is located in a housing, and the housing is composed of a card cover 1 and a card base 3 by ultrasonic welding, buckles or glue bonding. 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 placing the immunoassay test strip 2. In some cases, the card cover 1 is provided with a plurality of buckles extending downward (not shown in the figure), and the card base 3 is provided with a plurality of card slots 31 extending upward. The buckles provided on the card cover 1 and the card 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 is fixed in the test paper slot 32. In some cases, the multiple buckles on the card cover 1 are symmetrically distributed on both sides of the card cover 1, and the multiple card slots 31 on the card base 3 are symmetrically distributed on both sides of the card base 3.

[0023] 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, and under the action of capillaries, the sample migrates to the sample absorption pad 26 along the length direction of the immunoassay test strip 2. The observation window 12 is arranged above the detection line 23 and the control line 24 of the detection pad 25. The excitation light emitted by the external light source can be irradiated onto the detection 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 detection line 23 and the control line 24 can also be output to the detector through the observation window 12.

[0024] During the test, 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 inserted into the analyzer for testing after a period of reaction outside the analyzer, or the clinical sample is immediately inserted into the analyzer after the test card is added, and then tested after a period of reaction in the analyzer. The analyzer used in the present invention can be a commercially available analyzer, such as a dry fluorescent immunoassay analyzer FIC-Q100N (Suzhou Hemai Precision Instrument Co., Ltd.).

[0025] Depending on the different analytes to be tested (such as antigens, antibodies or haptens) and the different immunoassay principles, the substances coated on the label pad 22 and the detection line will vary. Here, the analyte to be tested is TT3 and the detection principle is the competitive method as an example for explanation. The label pad 22 is coated with T3 antibodies labeled with fluorescent markers and rabbit IgG antibodies labeled with fluorescent markers, the detection line 23 is coated with a conjugate of T3 antigen and carrier protein BSA, and the control line 24 is coated with goat anti-rabbit IgG antibodies. In this way, when the clinical sample treated with a dissociating agent in advance is added to the sample pad 21 through the sample addition port 11, the clinical sample carries the T3 antibodies labeled with fluorescent markers and the rabbit IgG antibodies labeled with fluorescent markers and flows along the length direction of the immunoassay test strip 2. When the clinical sample reaches the label pad 22, the fluorescent marker-labeled T3 antibody specifically binds to T3 (if present) in the clinical sample, and the formed fluorescent marker-T3 antibody-T3 complex continues to flow, and the T3-BSA conjugate coated on the detection line 23 competes with T3 in the clinical sample for the fluorescent marker-labeled T3 antibody, and the detection signal can be generated after the excitation light emitted by the light source in the analyzer illuminates the detection line 23. When flowing to the control line 24, the fluorescent marker-labeled rabbit IgG antibody specifically binds to the goat anti-rabbit IgG antibody coated on the control line 24 to form a complex fixed on the control line 24, and the control signal can be generated after the excitation light emitted by the light source in the analyzer illuminates the control line 24.

[0026] 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 rabbit IgG antibody labeled with a fluorescent marker can also be replaced by colored colloidal particles such as latex microspheres, colloidal gold, colloidal carbon, colloidal selenium, etc.

[0027] 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 inside or on the 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 have europium chelates embedded inside and carry carboxyl groups on the surface (Suzhou Weidu Biotechnology Co., Ltd., product number FT0200CA), with an excitation wavelength of 360nm and an emission wavelength of 615nm.

[0028] The fluorescent marker used in the present invention can also be a fluorescent compound, which 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.

[0029] The fluorescent marker used in the present invention can also be a colored fluorescent microsphere, that is, a microsphere or microbead with a fluorescent compound without a light-emitting delay characteristic wrapped on the surface or inside, which can generate a fluorescent signal without a light-emitting delay characteristic when irradiated with an excitation light of a suitable wavelength. The colored luminescent microsphere can be selected from green fluorescent microspheres, blue fluorescent microspheres, red fluorescent microspheres, yellow fluorescent microspheres and colored fluorescent microspheres (emitting fluorescence of multiple specific colors).

[0030] Whether in time-resolved fluorescent microspheres or in 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, polydivinyl benzene, 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 and other groups, 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 20nm to 100μm; the particle size of the colored fluorescent microspheres is 100nm to 100μm.

[0031] 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.

[0032] Example 1: Dissociation agent formulation

[0033] 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 pH 7.1-7.4.

[0034] Formulation 1: 0.1 g elastase, 1.0 g DTT, 2.0 g 5-bromo-2-methylpyridine, Tween-40 0.5% (v / v), and buffer 10 mM PBS (pH 7.1-7.4) in 1 L deionized water.

[0035] Formulation 2: 1.0 g elastase, 3.0 g DTT, 0.1 g 5-bromo-2-methylpyridine, Tween-40 0.5% (v / v), and 10 mM PBS buffer (pH 7.1-7.4) in 1 L deionized water.

[0036] Formulation 3: 2.0 g elastase, 0.1 g DTT, 1.0 g 3-bromo-5-hydroxypyridine, Tween-40 2% (v / v), and buffer 10 mM PBS (pH 7.1-7.4) in 1 L deionized water.

[0037] In addition, the 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).

[0038] In order to maintain the stability of the dissociator formula during storage, a preservative can be added to the dissociator formula. The preservative can be selected from sodium azide, thimerosal, sodium benzoate, Proclin-150, Proclin-200, Proclin-300 and Proclin-5000, and its concentration can be selected to be 0.1% to 2% (v / v).

[0039] Example 2: Dissociation Agent Preparation Method

[0040] A method for preparing a dissociation agent for detecting TT3 and TT4 in human serum comprises the following steps:

[0041] 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, and then add the surfactant and stir to mix after it is completely mixed and dissolved.

[0042] Example 3: TT3 performance test

[0043] A total of 30 clinical serum samples (including low, medium and high values) were collected using the total triiodothyronine assay kit (chemiluminescent microparticle immunoassay) produced by Abbott (National Medical Device Injection No. 20142405914) and the Abbott immunoassay analyzer ARCHITECT i2000, and the samples were numbered 1 to 30. For each sample, 40 μL of the sample was taken with a pipette into 5 sample reaction tubes, and the reaction tubes were numbered A, B, C, D, E plus the sample number, that is, A1-A30, B1-B30, C1-C30, D1-D30, E1-E30.

[0044] Add 100 μL of the dissociation agent formula 1 in Example 1 to each reaction tube in group A;

[0045] Add 100 μL of the dissociation agent formula 2 in Example 1 to each reaction tube in group B;

[0046] Add 100 μL of dissociation agent formula 3 in Example 1 to each reaction tube in group C;

[0047] Add 100 μL of the control dissociation agent formula to each reaction tube in group D. The specific formula is as follows:

[0048] In 1L deionized water, 0.5g EDTA, 2.5g 5-bromo-2-methylpyridine, 2.5g 3-bromo-5-hydroxypyridine, 1ml Proclin-300, 5ml Tween-40, 10g ANS, and 1g sodium citrate.

[0049] Add 100 μL of conventional treatment solution to each reaction tube in group E. The specific formula is as follows:

[0050] 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.

[0051] After mixing the above reaction tube solutions, stand at room temperature for 10 minutes, and then use the total triiodothyronine (T3) quantitative detection kit (fluorescence immunoassay) reagent (catalog number F131-20211) produced by Aikang Biotechnology (Hangzhou) Co., Ltd. in conjunction with the dry fluorescence immunoassay instrument (FIC-Q100N) produced by Suzhou Hemai Precision Instrument Co., Ltd. for detection. 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 chemiluminescence detection value). The results are shown in Table 1.

[0052] Table 1 TT3 comparative experimental results (unit: nmol / L)

[0053]

[0054]

[0055] From the analysis of the comparison results in Table 1, the conclusions are as follows:

[0056] 1. After treating clinical serum samples with conventional treatment solution (Group E), the measured results are generally much lower. This is because most of the TT3 in the clinical samples has not been dissociated and cannot be bound by the paired antibodies. For example, the detection value of Abbott chemiluminescence reagent for sample No. 30 is 9.84nmol / L, the detection value of Group A is 9.11nmol / L, the detection value of Group B is 9.45nmol / L, the detection value of Group C is 9.26nmol / L, and the detection value of Group E is only 1.35nmol / L.

[0057] 2. After the clinical samples were treated with the treatment solution containing the dissociation agent ANS (Group D), the detection values ​​of the clinical samples were generally greater than those of the clinical samples 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 large, which leads to abnormal fluorescence signal values ​​of clinical samples and a large deviation in the overall detection concentration. In terms of clinical correlation with the detection value of Abbott 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.

[0058] 3. The detection value of the AC group after the dissociation agent treated the clinical samples had a good correlation with the detection value of the Abbott chemiluminescence reagent, both of which were greater than 0.95. This is because the dissociation agent prepared by the AC group can fully dissociate TT3 in the clinical samples, and the dissociation agent itself will not affect the detection of the fluorescence signal value.

[0059] Example 4: TT4 performance test

[0060] A total of 30 clinical serum samples (including low, medium and high values) were collected using the total thyroxine assay kit (chemiluminescent microparticle immunoassay) produced by Abbott (National Medical Device Injection No. 20162404093) and the instrument model (ARCHITECT i2000), and the samples were numbered 1 to 30. For each sample, 40 μL of the sample was taken with a pipette into 5 sample reaction tubes, which were numbered A, B, C, D, and E, plus the sample number, namely A1-A30, B1-B30, C1-C30, D1-D30, and E1-E30.

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

[0062] Add 100 μL of the dissociation agent formula 2 in Example 1 to each reaction tube in group B;

[0063] Add 100 μL of dissociation agent formula 3 in Example 1 to each reaction tube in group C;

[0064] Add 100 μL of the control dissociation agent formula to each reaction tube in group D. The specific formula is as follows:

[0065] In 1L deionized water, 0.5g EDTA, 2.5g 5-bromo-2-methylpyridine, 2.5g 3-bromo-5-hydroxypyridine, 1ml Proclin-300, 5ml Tween-40, 10g ANS, and 1g sodium citrate.

[0066] Add 100 μL of conventional treatment solution to each reaction tube in group E. The specific formula is as follows:

[0067] 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.

[0068] After mixing the above reaction tube solutions, stand at room temperature for 10 minutes, and then use the total thyroxine (T4) quantitative detection kit (fluorescence immunoassay) reagent registered by Aikang Biotechnology (Hangzhou) Co., Ltd. (Cat. No. F131-202311) in conjunction with the dry fluorescence immunoassay instrument (FIC-Q100N) produced by Suzhou Hemai Precision Instrument Co., Ltd. for detection. 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 instrument ARCHITECT i2000 (hereinafter referred to as Abbott's chemiluminescence detection value), and the results are shown in Table 2.

[0069] Table 2 TT4 comparative experimental results (unit: nmol / L)

[0070]

[0071]

[0072] From the analysis of the comparison results in Table 2, the conclusions are as follows:

[0073] 1. After treating clinical serum samples with conventional treatment solution (Group E), the test results are generally lower than normal. This is because most of the TT4 in the clinical samples has not been dissociated and cannot be bound by the paired antibodies. For example, the detection value of Abbott chemiluminescence reagent for sample No. 26 was 196.82nmol / L, the detection value of Group A was 215.00nmol / L, the detection value of Group B was 195.65nmol / L, the detection value of Group C was 231.70nmol / L, and the detection value of Group E was only 1.72nmol / L.

[0074] 2. After the samples were treated with the treatment solution containing the dissociation agent ANS (Group D), the measured values ​​of the clinical samples were generally greater than the measured values ​​of the clinical samples 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 large, which leads to abnormal fluorescence signal values ​​of the sample detection and a large deviation in the overall detection concentration. In terms of clinical correlation with the detection value of Abbott chemiluminescence reagent, the correlations of Groups AC were 0.9819, 0.9797 and 0.9778, respectively, while the correlation of Group D was only 0.7796.

[0075] 2. After the clinical samples were treated with the treatment solution containing the dissociation agent ANS (Group D), the detection values ​​of the clinical samples were generally greater than those of the clinical samples 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 large, which leads to abnormal fluorescence signal values ​​of clinical samples and a large deviation in the overall detection concentration. In terms of clinical correlation with the detection value of Abbott 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.

[0076] 3. The detection value of the AC group after the dissociation agent treated the clinical samples had a good correlation with the detection value of the Abbott chemiluminescence reagent, both of which were greater than 0.95. This is because the dissociation agent prepared by the AC group can fully dissociate TT4 in the clinical samples, and the dissociation agent itself will not affect the detection of the fluorescence signal value.

[0077] Example 5: Reducing agent evaluation experiment

[0078] Formulation 4: 0.1 g elastase, 1.0 g TCEP-HCl, 2.0 g 5-bromo-2-methylpyridine, Tween-40 0.5% (v / v), and buffer 10 mM PBS (pH 7.1-7.4) in 1 L deionized water.

[0079] Formulation 5: 0.1 g elastase, 3.0 g TCEP-HCl, 2.0 g 5-bromo-2-methylpyridine, Tween-40 0.5% (v / v), and buffer 10 mM PBS (pH 7.1-7.4) in 1 L deionized water.

[0080] 15 fresh clinical blood samples were collected from Abbott's total triiodothyronine assay kit (chemiluminescent microparticle immunoassay) (National Medical Device Injection No. 20142405914) and matched with Abbott's immunoanalyzer ARCHITECT i2000, and the concentration distribution was required to be uniform. Then the TT3 performance test method in Implementation 3 was used for testing, except that only three reaction tubes were selected, and 100 μL of dissociation agent formula 1 in Example 1 was added to the reaction tube of Group A, 100 μL of dissociation agent formula 4 was added to the reaction tube of Group B, and 100 μL of dissociation agent formula 5 was added to the reaction tube of Group B. The results are shown in Table 3.

[0081] Table 3 Reducing agent evaluation test results (unit: nmol / L)

[0082]

[0083] It can be seen from Table 3 that after selecting dissociation agent formula 1, formula 4 and formula 5, the correlation coefficients between the concentration values ​​calculated by fluorescent immunoassay and the Abbott chemiluminescence detection values ​​are all greater than 0.95, which means that the dissociation agent formula 1, formula 4 and formula 5 can fully dissociate TT3 in the sample, so that the TT3 value in the detected sample is more accurate.

[0084] 15 fresh clinical blood samples were collected with the total thyroxine assay kit (chemiluminescent microparticle immunoassay) (National Medical Device Injection No. 20162404093) produced by Abbott Laboratories and the instrument model (ARCHITECT i2000) with uniform concentration distribution. Then the TT4 performance test method in Implementation 4 was used for testing, except that only three reaction tubes were selected, and 100 μL of dissociation agent formula 1 in Example 1 was added to the reaction tube of Group A, 100 μL of dissociation agent formula 4 was added to the reaction tube of Group B, and 100 μL of dissociation agent formula 5 was added to the reaction tube of Group B. The results are shown in Table 4.

[0085] Table 4 Reducing agent evaluation test results (unit: nmol / L)

[0086]

[0087] It can be seen from Table 4 that after selecting dissociation agent formula 1, formula 4 and formula 5, the correlation coefficients between the concentration values ​​calculated by fluorescent immunoassay and the Abbott chemiluminescence detection values ​​are all greater than 0.95, which means that the dissociation agent formula 1, formula 4 and formula 5 can fully dissociate TT4 in the sample, so that the TT4 value in the detected sample is more accurate.

[0088] Example 6: Surfactant Evaluation Experiment

[0089] Formulation 6: 0.1 g elastase, 1.0 g DTT, 2.0 g 5-bromo-2-methylpyridine, Tween-20 0.5% (v / v), and buffer 10 mM PBS (pH 7.1-7.4) in 1 L deionized water.

[0090] Formulation 7: 0.1 g elastase, 1.0 g DTT, 2.0 g 5-bromo-2-methylpyridine, Triton X-100 0.5% (v / v), and buffer 10 mM PBS (pH 7.1-7.4) in 1 L deionized water.

[0091] 15 fresh clinical blood samples were collected from Abbott's total triiodothyronine assay kit (chemiluminescent microparticle immunoassay) (National Medical Device Injection No. 20142405914) and matched with Abbott's immunoassay analyzer ARCHITECT i2000, and the concentration distribution was required to be uniform. Then, the TT3 performance test method was implemented in accordance with the three methods, except that only three reaction tubes were selected, and 100 μL of dissociation agent formula 1 in Example 1 was added to the reaction tube of group A, 100 μL of dissociation agent formula 6 was added to the reaction tube of group B, and 100 μL of dissociation agent formula 7 was added to the reaction tube of group B. The results are shown in Table 5.

[0092] Table 5 Surfactant evaluation test results (unit: nmol / L)

[0093]

[0094] It can be seen from Table 5 that after selecting dissociation agent formula 1, formula 6 and formula 7, the correlation coefficients between the concentration values ​​calculated by fluorescent immunoassay and the Abbott chemiluminescence detection values ​​are all greater than 0.95, which means that the dissociation agent formula 1, formula 6 and formula 7 can fully dissociate TT3 in the sample, so that the TT3 value in the detected sample is more accurate.

[0095] 15 fresh clinical blood samples were collected with the total thyroxine assay kit (chemiluminescent microparticle immunoassay) (National Medical Device Injection No. 20162404093) produced by Abbott Laboratories and the instrument model (ARCHITECT i2000) with uniform concentration distribution. Then the TT4 performance test method in Implementation 4 was used for testing, except that only three reaction tubes were selected, and 100 μL of dissociation agent formula 1 in Example 1 was added to the reaction tube of Group A, 100 μL of dissociation agent formula 6 was added to the reaction tube of Group B, and 100 μL of dissociation agent formula 7 was added to the reaction tube of Group B. The results are shown in Table 6.

[0096] Table 6 Surfactant evaluation test results (unit: nmol / L)

[0097]

[0098] It can be seen from Table 6 that after selecting dissociation agent formula 1, formula 4 and formula 5, the correlation coefficients between the concentration values ​​calculated by fluorescent immunoassay and the Abbott chemiluminescence detection values ​​are all greater than 0.95, which means that dissociation agent formula 1, formula 6 and formula 7 can fully dissociate TT4 in the sample, so that the TT4 value in the detected sample is more accurate.

[0099] Example 7: Buffer evaluation experiment

[0100] Formulation 8: 0.1 g elastase, 1.0 g DTT, 2.0 g 5-bromo-2-methylpyridine, Tween-40 1.0% (v / v), and buffer 50 mM Tris-HCl (pH 7.1-7.4) in 1 L deionized water.

[0101] 15 fresh clinical blood samples were collected from Abbott's total triiodothyronine assay kit (chemiluminescent microparticle immunoassay) (National Medical Device Injection No. 20142405914) and matched with Abbott's immunoassay analyzer ARCHITECT i2000, and the concentration distribution was required to be uniform. Then the TT3 performance test method in Implementation 3 was used for testing, except that only two reaction tubes were selected, and 100 μL of dissociation agent formula 1 in Example 1 was added to the reaction tube of Group A, and 100 μL of dissociation agent formula 8 was added to the reaction tube of Group B. The results are shown in Table 7.

[0102] Table 7 Buffer evaluation test results (unit: nmol / L)

[0103]

[0104]

[0105] It can be seen from Table 7 that after selecting dissociation agent formula 1 and formula 8, the correlation coefficients between the concentration values ​​calculated by fluorescent immunoassay and the Abbott chemiluminescence detection values ​​are both greater than 0.95, which indicates that dissociation agent formula 1 and formula 8 can fully dissociate TT3 in the sample, thereby making the detected TT3 value in the sample more accurate.

[0106] 15 fresh clinical blood samples were collected with the total thyroxine assay kit (chemiluminescent microparticle immunoassay) (National Medical Device Injection No. 20162404093) produced by Abbott Laboratories and the instrument model (ARCHITECT i2000) with uniform concentration distribution. Then, the TT4 performance test method in Implementation 4 was used for testing, except that only three reaction tubes were selected, and 100 μL of dissociation agent formula 1 in Example 1 was added to the reaction tube of Group A, and 100 μL of dissociation agent formula 8 was added to the reaction tube of Group B. The results are shown in Table 8.

[0107] Table 8 Buffer evaluation test results (unit: nmol / L)

[0108]

[0109]

[0110] It can be seen from Table 8 that after selecting dissociation agent formula 1 and formula 8, the correlation coefficients between the concentration values ​​calculated by fluorescent immunoassay and the Abbott chemiluminescence detection values ​​are both greater than 0.95, which indicates that dissociation agent formula 1 and formula 8 can fully dissociate TT4 in the sample, thereby making the detected TT4 value in the sample more accurate.

[0111] Example 8: Dissociation Agent Evaluation Experiment

[0112] Formulation 9: 0.1 g elastase, Tween-40 0.5% (v / v), buffer 10 mM PBS (pH 7.1-7.4) in 1 L deionized water;

[0113] Formulation 10: 1.0 g elastase, Tween-40 0.5% (v / v), buffer 10 mM PBS (pH 7.1-7.4) in 1 L deionized water;

[0114] Formulation 11: 0.1 g elastase, 0.1 g DTT, Tween-40 0.5% (v / v), buffer 10 mM PBS (pH 7.1-7.4) in 1 L deionized water;

[0115] Formulation 12: 0.1 g elastase, 1.0 g DTT, Tween-40 0.5% (v / v), buffer 10 mM PBS (pH 7.1-7.4) in 1 L deionized water;

[0116] Formulation 13: 1.0 g elastase, 0.1 g DTT, Tween-40 0.5% (v / v), buffer 10 mM PBS (pH 7.1-7.4) in 1 L deionized water;

[0117] Formulation 14: 1.0 g elastase, 1.0 g DTT, Tween-40 0.5% (v / v), buffer 10 mM PBS (pH 7.1-7.4) in 1 L deionized water.

[0118] Collect 5 cases of fresh clinical blood samples of Abbott's total triiodothyronine assay kit (chemiluminescent microparticle immunoassay) (National Medical Device Injection 20142405914) with Abbott's immunoassay analyzer ARCHITECT i2000, and require uniform concentration distribution. Then, the TT3 performance test method in Implementation 3 was used for detection, except that only 6 reaction tubes were selected, and 100 μL of dissociation agent formulas 9, 10, 11, 12, 13 and 14 were added to the reaction tubes of 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 of group G. The results are shown in Table 9.

[0119] Table 9 Dissociation agent evaluation test results (unit: nmol / L)

[0120]

[0121]

[0122] It can be seen from Table 9 that, 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.

[0123] Collect 5 fresh clinical blood samples of total thyroxine assay kit (chemiluminescent microparticle immunoassay) (National Medical Device Injection No. 20162404093) produced by Abbott Laboratories and the instrument model (ARCHITECT i2000) with uniform concentration distribution. Then, the TT4 performance test method in Implementation 3 was used for testing, except that only 6 reaction tubes were selected, and 100 μL of dissociation agent formulas 9, 10, 11, 12, 13 and 14 were added to the reaction tubes of 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 of group G. The results are shown in Table 10.

[0124] Table 10 Dissociation agent evaluation test results (unit: nmol / L)

[0125]

[0126] It can be seen from Table 10 that, 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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