Latex immunoturbidimetric detection reagent, kit and detection method

Abstract

The application provides a latex immunoturbidimetric detection reagent, a kit and a detection method. The latex immunoturbidimetric detection reagent comprises a buffer and an ionic liquid, the ionic liquid comprises an ionic liquid containing an imidazole structure, the ionic liquid containing the imidazole structure comprises 1-alkyl-3-methyl imidazole halide or 1-alkyl-3-methyl imidazole fluoride, the halogen in the 1-alkyl-3-methyl imidazole halide comprises chlorine, bromine or iodine, and the alkyl in the 1-alkyl-3-methyl imidazole halide or 1-alkyl-3-methyl imidazole fluoride comprises alkyl containing 2, 3, 4, 5, 6 or 7 C atoms. The application can solve the problem of a small linear range in the prior art latex turbidimetric method, and is suitable for the field of immunoturbidimetric detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunoturbidimetry, in particular to a latex immunoturbidimetry detection reagent, kit and detection method. BACKGROUND

[0002] In the field of in vitro diagnosis, latex immunoturbidimetry is a method for detecting immune reactions. The basic principle of latex immunoturbidimetry is that after the surface of latex particles of a certain particle size is cross-linked with monoclonal or polyclonal antibodies or antigens, the measured antigen or antibody reacts with the microsphere conjugate to form an antigen-antibody-microsphere complex, which causes the reagent to appear turbidity, and the change in turbidity can be detected and recorded by a turbidimeter or a light density measuring instrument. Generally, when the concentration or amount of the target substance is high, the number of antigen-antibody-microsphere complexes also increases, causing more particles in the reagent system to aggregate or settle, thereby increasing the turbidity of the solution. According to the change in turbidity, the concentration or amount of the target substance can be determined by comparing the turbidity of the sample to be measured with the turbidity of a standard curve or a control sample of known concentration.

[0003] In recent years, with the market demand, the linear range of many latex immunoturbidimetry reagents is required to be higher and higher, but the existing immunoturbidimetry reagents are difficult to accurately detect high-concentration samples. For example, retinol binding protein (RBP), the serum RBP level is closely related to human kidney function. When kidney disease occurs, due to the decrease in glomerular filtration rate and the dysfunction of renal tubular reabsorption, the RBP in serum and urine is significantly increased, and the concentration can be as high as 200 mg / L or more. The conventional RBP measurement range is 3-120 mg / L, therefore, RBP determination reagents with high linearity can better meet the clinical needs. For example, anti-streptolysin O (ASO) project, when diseases such as rheumatic fever, acute glomerulonephritis, erysipelas, and streptococcal infection occur, ASO will increase significantly. In patients with rheumatic fever, the level of anti-streptolysin O will increase significantly, and some patients with rheumatic fever will even increase to 1200 IU / mL or more. The linearity of most anti-streptolysin O determination reagent kits (latex immunoturbidimetry) on the market can only reach about 800 IU / mL, and when high values are encountered, Hook effect will occur, affecting the judgment of the results, and cannot meet the clinical needs. SUMMARY

[0004] The main purpose of the present application is to provide a latex immunoturbidimetry detection reagent, kit and detection method to solve the problem of narrow linear range of latex turbidimetry determination in the prior art.

[0005] In order to achieve the above-mentioned purpose, according to a first aspect of the present application, a latex immunoturbidimetric detection reagent is provided, the detection reagent comprising a buffer and an ionic liquid, the ionic liquid comprising an ionic liquid containing an imidazole structure, the ionic liquid containing an imidazole structure comprising 1-alkyl-3-methyl imidazole halide or 1-alkyl-3-methyl imidazole fluoride, the alkyl in the 1-alkyl-3-methyl imidazole halide or 1-alkyl-3-methyl imidazole fluoride comprising an alkyl group consisting of 2, 3, 4, 5, 6 or 7 C atoms, the halogen in the 1-alkyl-3-methyl imidazole halide comprising chlorine, bromine or iodine.

[0006] Further, the 1-alkyl-3-methyl imidazole halide comprises one or more of 1-butyl-3-methyl imidazole bromide, 1-ethyl-3-methyl imidazole bromide, 1-hexyl-3-methyl imidazole chloride, 1-hexyl-3-methyl imidazole bromide or 1-heptyl-3-methyl imidazole bromide;

[0007] Preferably, the 1-alkyl-3-methyl imidazole fluoride is 1-alkyl-3-methyl imidazole tetrafluoroborate, more preferably, the 1-alkyl-3-methyl imidazole tetrafluoroborate comprises 1-butyl-3-methyl imidazole tetrafluoroborate or 1-hexyl-3-methyl imidazole tetrafluoroborate; preferably, the ionic liquid comprises 1-hexyl-3-methyl imidazole chloride; preferably, the mass content of the 1-hexyl-3-methyl imidazole chloride in the detection reagent is 5%-10%.

[0008] Further, the buffer comprises any one of Tris-HCl buffer, Na2HPO4-NaH2PO4 buffer, disodium hydrogen phosphate-citric acid buffer, glyoxal-HCl buffer and barbital buffer; preferably, the pH value of the buffer is 5.0-10.0; preferably, the buffer comprises Tris-HCl buffer with a pH value of 7.0-9.0 and a concentration of 50-500 mmol / L.

[0009] Further, the buffer further comprises one or more of inorganic salt, polyethylene glycol, ion chelating agent or surfactant; preferably, the inorganic salt comprises one or more of sodium chloride, potassium chloride, sodium acetate or magnesium chloride; preferably, the concentration of the inorganic salt is 5-10 g / L; preferably, the ion chelating agent is any one of EDTA-2Na, EDTA-4Na, potassium sodium tartrate and ammonium citrate; preferably, the polyethylene glycol comprises one or more of PEG800, PEG2000, PEG6000, PEG8000, PEG10000 or PEG20000; preferably, the surfactant comprises one or more of Tween 20, Tween 80, Tritin X100, Brij35, Brij58 or Span80.

[0010] In order to achieve the above-mentioned purpose, according to a second aspect of the present application, a latex immunoturbidimetric kit is provided, the kit comprising a first reagent, the first reagent being the above-mentioned detection reagent.

[0011] Further, the kit further comprises a second reagent; the second reagent comprises an antibody or an antigen-coated latex microsphere; preferably, in the kit, the first reagent and the second reagent are independently provided; preferably, the kit further comprises a calibrator, the calibrator comprising a corresponding antigen or antibody, and a human serum matrix, a preservative and a stabilizer; preferably, the preservative comprises one or more of sodium azide, ProClin 300, ProClin 150, ProClin 950, gentamicin, penicillin, BiT, potassium sorbate or sodium benzoate; preferably, the stabilizer comprises one or more of bovine serum albumin, trehalose, mannitol, ethylene glycol, glycerol or casein; preferably, the antibody comprises a retinol antibody or a ferritin antibody; preferably, the antigen comprises a streptolysin O antigen.

[0012] Further, the particle size of the latex microsphere is 80-300 nm; preferably, in the kit containing the retinol antibody, the particle size of the latex microsphere is 80-180 nm, more preferably 80-120 nm; preferably, in the kit containing the streptolysin O antigen, the particle size of the latex microsphere is 100-250 nm, more preferably 100-150 nm; preferably, in the kit containing the ferritin antibody, the particle size of the latex microsphere is 200-300 nm, more preferably 220-250 nm; preferably, the latex microspheres have the same specification.

[0013] In order to achieve the above-mentioned purpose, according to a third aspect of the present application, a latex immunoturbidimetric detection method for a protein to be detected is provided, the method comprising: mixing a sample containing the protein to be detected with a first reagent to obtain a sample ionic liquid mixture; mixing the sample ionic liquid mixture with a second reagent to perform latex immunoturbidimetric detection, and detecting the concentration of the protein to be detected; wherein the first reagent comprises an ionic liquid, the ionic liquid comprising an imidazole-containing ionic liquid, the imidazole-containing ionic liquid comprising a 1-alkyl-3-methyl imidazole halide or a 1-alkyl-3-methyl imidazole fluoride, the alkyl in the 1-alkyl-3-methyl imidazole halide or the 1-alkyl-3-methyl imidazole fluoride comprising an alkyl group consisting of 2, 3, 4, 5, 6 or 7 C atoms, the halogen in the 1-alkyl-3-methyl imidazole halide comprising chlorine, bromine or iodine; the second reagent comprising an antigen or an antibody-coated latex microsphere.

[0014] Further, the protein to be detected includes retinol binding protein, anti-streptolysin O or ferritin; preferably, for detection of retinol binding protein, the second reagent includes latex microspheres coated with retinol antibody; preferably, for detection of anti-streptolysin O, the second reagent includes latex microspheres coated with streptolysin antigen O; preferably, for detection of ferritin, the second reagent includes latex microspheres coated with ferritin antibody; preferably, the concentration of retinol binding protein is 0-300 mg / L, more preferably 100-300 mg / L; preferably, the concentration of anti-streptolysin O is 0-1500 IU / mL, more preferably 720-1500 IU / mL; preferably, the concentration of ferritin is 0-1000 mg / 100 mL; preferably, the particle size of the latex microspheres is 80-300 nm; preferably, in the kit containing retinol antibody, the particle size of the latex microspheres is 80-180 nm, more preferably 80-120 nm; preferably, in the kit containing streptolysin antigen O, the particle size of the latex microspheres is 100-250 nm, more preferably 100-150 nm; preferably, in the kit containing ferritin antibody, the particle size of the latex microspheres is 200-300 nm, more preferably 220-250 nm; preferably, the specifications of the latex microspheres are the same.

[0015] Further, the 1-alkyl-3-methyl imidazole halide salt includes one or more of 1-butyl-3-methyl imidazole bromide, 1-ethyl-3-methyl imidazole bromide, 1-hexyl-3-methyl imidazole chloride, 1-hexyl-3-methyl imidazole bromide or 1-heptyl-3-methyl imidazole bromide; preferably, the 1-alkyl-3-methyl imidazole fluoride is 1-alkyl-3-methyl imidazole tetrafluoroborate; more preferably, the 1-alkyl-3-methyl imidazole tetrafluoroborate includes 1-butyl-3-methyl imidazole tetrafluoroborate or 1-hexyl-3-methyl imidazole tetrafluoroborate; preferably, the ionic liquid includes 1-hexyl-3-methyl imidazole chloride; preferably, the mass content of the 1-hexyl-3-methyl imidazole chloride in the first reagent is 5%-10%; preferably, the first reagent further includes a buffer solution, and the buffer solution includes any one of Tris-HCl buffer solution, Na2HPO4-NaH2PO4 buffer solution, disodium hydrogen phosphate-citric acid buffer solution, glyoxal-HCl buffer solution and barbiturate buffer solution; preferably, the pH value of the buffer solution is 5.0-10.0; preferably, the buffer solution includes Tris-HCl buffer solution with a pH value of 7.0-9.0 and a concentration of 50-500 mmol / L; preferably, the buffer solution further includes one or more of an inorganic salt, a polyethylene glycol, an ion chelating agent or a surfactant; preferably, the inorganic salt includes one or more of sodium chloride, potassium chloride, sodium acetate or magnesium chloride; preferably, the concentration of the inorganic salt is 5-10 g / L; preferably, the ion chelating agent is any one of EDTA-2Na, EDTA-4Na, potassium sodium tartrate and ammonium citrate; preferably, the polyethylene glycol includes one or more of PEG800, PEG2000, PEG6000, PEG8000, PEG10000 or PEG20000; preferably, the surfactant includes one or more of Tween 20, Tween 80, Tritin X100, Brij35, Brij58 or Span80.

[0016] By using the above-mentioned detection reagent, the linear range in the latex immunoturbidimetry can be improved, and the ionic liquid in the above-mentioned detection reagent can ensure the detection accuracy and precision, and realize the detection of higher concentration samples. BRIEF DESCRIPTION OF DRAWINGS

[0017] The accompanying drawings, which form a part of the present application, are included to provide a further understanding of the application, and are incorporated herein for reference. The embodiments of the present application and its description are used to explain the present application and do not constitute an improper limitation to the present application. In the drawings:

[0018] Figure 1 A calibration reaction degree fitting curve result diagram of different ionic liquids according to Embodiment 1 of the present application is shown.

[0019] Figure 2 A calibration reaction rate fitting curve result map of different microsphere diameters according to Embodiment 1 of the present application is shown.

[0020] Figure 3 A calibration reaction rate fitting curve result map of different ionic liquid concentrations according to Embodiment 1 of the present application is shown.

[0021] Figure 4 A fitting result map of serum comparison tests according to Embodiment 1 of the present application is shown.

[0022] Figure 5 A calibration reaction rate fitting curve result map of different ionic liquid concentrations according to Embodiment 2 of the present application is shown.

[0023] Figure 6 A fitting result map of serum comparison tests according to Embodiment 2 of the present application is shown.

[0024] Figure 7 A calibration reaction rate fitting result map according to Embodiment 3 of the present application is shown.

[0025] Figure 8 A sample comparison fitting result map according to Embodiment 3 of the present application is shown. DETAILED DESCRIPTION

[0026] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below with reference to the embodiments.

[0027] Explanation of terms:

[0028] Linear range: refers to the change range of the concentration of the test substance in a linear form, in which the precision and accuracy of the test results obtained by using a method meet the requirements, and the interval between the maximum amount and the minimum amount. That is, the larger the linear range, the wider the concentration range that can be detected by the method.

[0029] Ionic liquid containing imidazole structure: refers to ionic liquid containing imidazole in the cation. The cation of the ionic liquid containing imidazole structure in the present application includes 1-alkyl-3-methyl imidazole.

[0030] As mentioned in the background, the latex immunoturbidimetric reagent in the prior art has poor discrimination for high-value samples, narrow detection linearity, and it is difficult to realize accurate detection of high-concentration samples, and it is difficult to meet the needs of immunodetection in the prior art.

[0031] The latex immunoturbidimetry generally has the characteristics of good specificity but poor sensitivity or good sensitivity but poor specificity. In the prior art, the lower and upper limits of linear detection are improved by using monoclonal antibodies, double antibodies and the like, but the price of the reagent such as the monoclonal antibody is high, which is not conducive to clinical popularization; the mixed cross-linking of large and small microspheres, the small particle size microspheres of about 60-100 nm, the reagent preparation process needs to use an ultrahigh speed centrifuge or a complex process such as tangential flow, which is also not conducive to the operation and popularization of the clinic.

[0032] Therefore, in the present application, the inventors attempt to develop a latex immunoturbidimetry detection reagent, which can realize accurate detection of high concentration samples, improve the linear range of immunoturbidimetry detection, and the cost of the detection reagent is low, the use method is simple, and the accurate detection of high concentration samples can be realized at low cost and simple method. Therefore, a series of protection schemes of the present application are proposed.

[0033] In a first typical embodiment of the present application, a latex immunoturbidimetry detection reagent is provided, which includes a buffer and an ionic liquid, the ionic liquid includes an imidazole-containing ionic liquid, the imidazole-containing ionic liquid includes 1-alkyl-3-methyl imidazole halide or 1-alkyl-3-methyl imidazole fluoride, the alkyl in the 1-alkyl-3-methyl imidazole halide or 1-alkyl-3-methyl imidazole fluoride includes an alkyl group composed of 2, 3, 4, 5, 6 or 7 C atoms, and the halogen in the 1-alkyl-3-methyl imidazole halide includes chlorine, bromine or iodine.

[0034] The ionic liquid is a compound composed of an organic cation and an organic / inorganic anion, which is in a liquid state at room temperature or near room temperature, and its physical properties depend on the structure of its anion and cation and the interaction between them, including electrostatic bonding, polarization, induction, dispersion force, hydrogen bond, dipole-dipole interaction, etc. The strong electrostatic interaction and the special hydrogen bond induced by the ionic liquid make the ionic liquid easy to form a special "ionic microenvironment". Due to the influence of electrostatic interaction, its force is much stronger than the hydrogen bond interaction of molecular system, so it shows unique performance in the application reaction system.

[0035] For conventional imidazole cation-based ionic liquids, the anion and cation form a hydrogen bond as a hydrogen bond acceptor and a hydrogen bond donor, respectively, and the electrostatic superposition leads to a hydrogen bond stronger than the intermolecular hydrogen bond, and a hydrogen bond network can be formed. The hydrogen bond under electrostatic interaction is the basic structural feature of this type of ionic liquid, which plays a dominant role in the formation of its performance.

[0036] The specific binding of antigen and antibody is mainly based on the complementarity of the molecular structure and stereo-configuration of antigen and antibody epitope (determinant), and the reversible immune reaction caused by the intermolecular attraction of hydrogen bond, hydrophobic and electrostatic interaction and van der Waals force. Under the condition of ion liquid-mediated immune reaction, on the one hand, due to the strong hydrogen bond force of ion liquid under the action of electrostatic interaction, the weak and reversible hydrogen bond bridge generated by the destruction of the close proximity of antibody and antigen makes some binding sites with insufficient affinity separate, to a certain extent, to inhibit the progress of immune reaction. On the other hand, the active sites of antigen and antibody are more relaxed, thereby improving the specificity of immune reaction.

[0037] The above detection reagent includes buffer and ionic liquid, which can be mixed with the sample, not only to provide suitable pH, ionic strength and other reaction conditions for subsequent antigen-antibody binding; and the ionic liquid in the above detection reagent can broaden the linear test range, reduce non-specific reaction and improve the accuracy of sample detection by inhibiting the progress of immune reaction.

[0038] The above alkyl group includes but is not limited to ethyl, propyl, butyl, pentyl, quaternary group or heptyl, preferably n-alkyl such as n-propyl, n-butyl, n-pentyl, n-quaternary group or n-heptyl. In a preferred embodiment, the 1-alkyl-3-methyl imidazole halide salt includes but is not limited to one or more of 1-butyl-3-methyl imidazole bromide ([Bmim]Br), 1-ethyl-3-methyl imidazole bromide ([Emim]Br), 1-hexyl-3-methyl imidazole chloride ([Hmim]Cl), 1-hexyl-3-methyl imidazole bromide ([Hmim]Br) or 1-heptyl-3-methyl imidazole bromide; preferably, the 1-alkyl-3-methyl imidazole fluoride is 1-alkyl-3-methyl imidazole tetrafluoroborate; more preferably, the 1-alkyl-3-methyl imidazole tetrafluoroborate includes 1-butyl-3-methyl imidazole tetrafluoroborate ([Bmim]BF4) or 1-hexyl-3-methyl imidazole tetrafluoroborate ([Hmim]BF4); preferably, the ionic liquid includes 1-hexyl-3-methyl imidazole chloride; preferably, the mass content of 1-hexyl-3-methyl imidazole chloride in the detection reagent is 5%-10%.

[0039] The above plurality of ionic liquids can improve the detection range of latex immune turbidimetry in the kit, preferably 1-hexyl-3-methyl imidazole chloride is selected as the ionic liquid, which can better improve the detection range as a component in the detection reagent, especially for the detection range of retinol binding protein or anti-streptolysin O.

[0040] In a preferred embodiment, the 1-alkyl-3-methyl imidazole halide salt comprises one or more of 1-butyl-3-methyl imidazole bromide, 1-ethyl-3-methyl imidazole bromide, 1-hexyl-3-methyl imidazole chloride, 1-hexyl-3-methyl imidazole bromide or 1-heptyl-3-methyl imidazole bromide; preferably, the 1-alkyl-3-methyl imidazole fluoride is 1-alkyl-3-methyl imidazole tetrafluoroborate; more preferably, the 1-alkyl-3-methyl imidazole tetrafluoroborate comprises 1-butyl-3-methyl imidazole tetrafluoroborate or 1-hexyl-3-methyl imidazole tetrafluoroborate; preferably, the ionic liquid comprises 1-hexyl-3-methyl imidazole chloride; preferably, the mass content of 1-hexyl-3-methyl imidazole chloride in the detection reagent is 5%-10%.

[0041] In a preferred embodiment, the buffer comprises any one of Tris-HCl buffer, Na2HPO4-NaH2PO4 buffer, disodium hydrogen phosphate-citric acid buffer, glyoxal-HCl buffer and barbiturate buffer; preferably, the pH value of the buffer is 5.0-10.0; preferably, the buffer comprises Tris-HCl buffer with pH 7.0-9.0 and 50-500 mmol / L.

[0042] In a preferred embodiment, the buffer further comprises one or more of inorganic salt, polyethylene glycol, ion chelating agent or surfactant; preferably, the inorganic salt comprises one or more of sodium chloride, potassium chloride, sodium acetate or magnesium chloride; preferably, the concentration of the inorganic salt is 5-10 g / L; preferably, the ion chelating agent is any one of EDTA-2Na, EDTA-4Na, potassium sodium tartrate and ammonium citrate; preferably, the polyethylene glycol comprises but is not limited to one or more of PEG800, PEG2000, PEG6000, PEG8000, PEG10000 or PEG20000; preferably, the surfactant comprises but is not limited to one or more of Tween20, Tween 80, Tritin X100, Brij35, Brij58 or Span80.

[0043] The buffer and inorganic salt in the above detection reagent are used to provide suitable pH, ionic strength and other reaction conditions for subsequent antigen-antibody binding, and the same components that can be used in such kits in the prior art can be flexibly replaced.

[0044] In a second typical embodiment of the present application, a latex immunoturbidimetric kit is provided, which comprises a first reagent, and the first reagent is the above detection reagent.

[0045] In a preferred embodiment, the kit further comprises a second reagent; the second reagent comprises an antibody or an antigen-coated latex microsphere; preferably, in the above-mentioned kit, the first reagent and the second reagent are independently provided; preferably, the kit further comprises a calibrator, the calibrator comprises a corresponding antigen or antibody, and a human serum matrix, a preservative, and a stabilizer; preferably, the preservative comprises one or more of, but is not limited to, sodium azide, ProClin 300, ProClin 150, ProClin 950, gentamicin, penicillin, BiT, potassium sorbate, or sodium benzoate; preferably, the stabilizer comprises one or more of, but is not limited to, bovine serum albumin, trehalose, mannitol, ethylene glycol, glycerol, or casein; preferably, the antibody comprises a retinol antibody or a ferritin antibody; preferably, the antigen comprises a streptolysin antigen O.

[0046] In the above-mentioned kit, by using the first reagent and the second reagent, the latex immunoturbidimetry method can be used to detect the concentration of different proteins, and due to the effect of the above-mentioned ionic liquid, the detection linearity of the kit is wider, and it is suitable for samples with high concentration of substances to be detected. The above-mentioned retinol antibody comprises a human retinol polyclonal antibody or a monoclonal antibody, and comprises an antibody derived from different animals, such as a goat anti-human retinol polyclonal antibody. Similarly, the above-mentioned ferritin antibody comprises, but is not limited to, a mouse anti-human ferritin polyclonal antibody. The above-mentioned kit can also comprise a calibrator, which contains an antigen or an antibody with the same content as the protein to be detected. When the kit is used to detect the protein concentration in the sample, the calibrator is used as a positive control group, and by using the determination data and the standard concentration of the calibrator, the protein concentration in the sample to be detected can be determined.

[0047] By using the kit in the present application, the latex microsphere immune analysis complex coated with high-sensitivity polyclonal antibodies for the immunoturbidimetry project is added with ionic liquid to inhibit the progress of the immune reaction, thereby widening the linear test range of the reagent. Without complex preparation process and without changing the test process. And by adding ionic liquid, the non-specific binding of antigen and antibody is reduced, the non-specific binding is reduced, and the accuracy of sample detection is improved. On the basis of widening the linear test range of the reagent, the sensitivity of the reagent detection is ensured. And the detection reagent of the present application does not depend on the components of high-cost monoclonal antibodies, double antibodies, and small-particle microspheres, the detection method is simple, and the production cost and detection cost can be reduced.

[0048] In a preferred embodiment, the latex microspheres have a particle size of 80-300 nm; preferably, in the kit containing the antibody to retinol, the latex microspheres have a particle size of 80-180 nm, more preferably 80-120 nm; preferably, in the kit containing the streptolysin O antigen, the latex microspheres have a particle size of 100-250 nm, more preferably 100-150 nm; preferably, in the kit containing the ferritin antibody, the latex microspheres have a particle size of 200-300 nm, more preferably 220-250 nm; preferably, the latex microspheres have the same size. The mass fraction of the latex microspheres in the antibody solution is 0.25-1 wt%. The particle size of the latex microspheres used in the above-mentioned kits includes but is not limited to 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 nm. The latex microspheres in the kit can be the same size or a mixture of different sizes, more preferably the same size and uniform particle size. Although due to the nature of the microspheres, not all microspheres of the same size (particle size) in commercially available or self-made microspheres are directly labeled with the size, but the particle size of the microspheres is distributed on both sides of the labeled size, and the proportion of microspheres within the specified particle size range reaches the specified value, i.e. meets the quality requirements (including but not limited to 90% of the microspheres have a particle size within ±30% of the labeled size). The above-mentioned "same size" refers to the use of microspheres of the same size (labeled size), rather than a mixture of two or more sizes. The above-mentioned limitation on the particle size of the latex microspheres is also a limitation on the size (labeled size) of the microspheres, such as the use of directly 80 nm latex microspheres, i.e. the size or labeled size of the latex microspheres is 80 nm, wherein the latex microspheres exist around 80 nm, rather than each microsphere in the used latex microspheres has a particle size greater than 80 nm. The particle size of the above-mentioned latex microspheres can be flexibly adjusted according to different components to be detected, so that the same size of latex microspheres is used in the kit to complete latex immunoturbidity.

[0049] In a third typical embodiment of the present application, an immunoturbidimetric method for detecting a protein to be detected is provided, which comprises: mixing a sample containing the protein to be detected with a first reagent to obtain a sample ionic liquid mixture; mixing the sample ionic liquid mixture with a second reagent to perform latex immunoturbidimetric detection, and detecting the concentration of the protein to be detected; wherein the first reagent comprises an ionic liquid, the ionic liquid comprises an ionic liquid containing an imidazole structure, the ionic liquid containing an imidazole structure comprises 1-alkyl-3-methyl imidazole halide or 1-alkyl-3-methyl imidazole fluoride, the alkyl in the 1-alkyl-3-methyl imidazole halide or 1-alkyl-3-methyl imidazole fluoride comprises an alkyl group consisting of 2, 3, 4, 5, 6 or 7 C atoms, and the halogen in the 1-alkyl-3-methyl imidazole halide comprises chlorine, bromine or iodine; the second reagent comprises antigen or antibody coated latex microspheres.

[0050] In the above immunoturbidimetric detection method, the protein to be detected is first mixed with the first reagent containing the ionic liquid, which not only provides suitable reaction conditions such as pH and ionic strength for subsequent antigen-antibody binding, but also the ionic liquid in the above detection reagent can broaden the linear test range, reduce non-specific reactions and improve the accuracy of sample detection by inhibiting the progress of the immune reaction. Further, the sample ionic liquid mixture obtained by mixing is mixed with the second reagent containing the antigen or antibody coated latex microspheres to realize the binding of the protein to be detected with the antigen or antibody.

[0051] In a preferred embodiment, the protein to be detected comprises retinol binding protein, anti-streptolysin O or ferritin; preferably, for the detection of retinol binding protein, the second reagent comprises retinol antibody coated latex microspheres; preferably, for the detection of anti-streptolysin O, the second reagent comprises streptolysin antigen O coated latex microspheres; preferably, for the detection of ferritin, the second reagent comprises ferritin antibody coated latex microspheres; preferably, the concentration of retinol binding protein is 0-300 mg / L, more preferably 100-300 mg / L; preferably, the concentration of anti-streptolysin O is 0-1500 IU / mL, more preferably 720-1500 IU / mL; preferably, the concentration of ferritin is 0-1000 mg / 100 mL; preferably, the particle size of the latex microspheres is 80-300 nm; preferably, in the kit containing retinol antibody, the particle size of the latex microspheres is 80-180 nm, more preferably 80-120 nm; preferably, in the kit containing streptolysin antigen O, the particle size of the latex microspheres is 100-250 nm, more preferably 100-150 nm; preferably, in the kit containing ferritin antibody, the particle size of the latex microspheres is 200-300 nm, more preferably 220-250 nm; preferably, the specifications of the latex microspheres are the same.

[0052] In a preferred embodiment, the 1-alkyl-3-methyl imidazole halide salt comprises one or more of 1-butyl-3-methyl imidazole bromide, 1-ethyl-3-methyl imidazole bromide, 1-hexyl-3-methyl imidazole chloride, 1-hexyl-3-methyl imidazole bromide or 1-heptyl-3-methyl imidazole bromide; preferably, the 1-alkyl-3-methyl imidazole fluoride is 1-alkyl-3-methyl imidazole tetrafluoroborate; more preferably, the 1-alkyl-3-methyl imidazole tetrafluoroborate comprises 1-butyl-3-methyl imidazole tetrafluoroborate or 1-hexyl-3-methyl imidazole tetrafluoroborate; preferably, the ionic liquid comprises 1-hexyl-3-methyl imidazole chloride; preferably, the mass content of 1-hexyl-3-methyl imidazole chloride in the first reagent is 5%-10%, including but not limited to 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10%; preferably, the first reagent further comprises a buffer, and the buffer comprises any one of Tris-HCl buffer, Na2HPO4-NaH2PO4 buffer, disodium hydrogen phosphate-citric acid buffer, glyoxal-HCl buffer and barbiturate buffer; preferably, the pH value of the buffer is 5.0-10.0, including but not limited to pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 or 10.0; preferably, the buffer comprises Tris-HCl buffer with pH 7.0-9.0 and 50-500 mmol / L; preferably, the buffer further comprises one or more of inorganic salt, polyethylene glycol, ion chelating agent or surfactant; preferably, the inorganic salt comprises one or more of sodium chloride, potassium chloride, sodium acetate or magnesium chloride; preferably, the concentration of the inorganic salt is 5-10 g / L; preferably, the ion chelating agent is any one of EDTA-2Na, EDTA-4Na, potassium sodium tartrate and ammonium citrate; preferably, the polyethylene glycol comprises one or more of PEG800, PEG2000, PEG6000, PEG8000, PEG10000 or PEG20000; preferably, the surfactant comprises one or more of Tween 20, Tween 80, Tritin X100, Brij 35, Brij 58 or Span 80.

[0053] By using the above-mentioned immunoturbidimetric detection method, the concentration of retinol binding protein can be detected by using latex turbidimetry. The linear range of this detection is wide, and accurate detection of high-concentration samples of 300 mg / L can be realized. The concentration of anti-streptolysin O can also be detected by using latex turbidimetry. The linear range of this detection is wide, and accurate detection of high-concentration samples of 1500 IU / mL can be realized. In addition, a part of non-specific immune reaction can be reduced, and the sample test is more accurate.

[0054] The beneficial effects of the present application will be further explained in detail below in connection with specific examples.

[0055] Example 1: Validation of retinol binding protein (RBP) project:

[0056] 1. Screening of ionic liquid:

[0057] Screening of ionic liquid types, and determining which factor in ionic liquid broadens the linear detection range: methyl imidazole, carbon chain length of cation, type of anion. Single factor test was used, and the scheme is shown in Table 1.

[0058] The components of the first reagent (R1) are as follows: the specific scheme is shown in Table 1.

[0059] The components of the second reagent (R2) are as follows: 0.5wt% goat anti-human retinol polyclonal antibody coated latex microspheres, pH 7.5 20mM Tris-HCl buffer, ethylenediaminetetraacetic acid disodium (EDTA-2Na) 5mM.

[0060] Table 1

[0061]

[0062]

[0063] Detection steps:

[0064] Detection instrument: full-automatic biochemical analyzer (model: Biossays C8).

[0065] Detection temperature: 37℃.

[0066] Detection wavelength: main wavelength: 570nm, secondary wavelength: 800nm.

[0067] Reaction time: 10 minutes, of which, incubation time 5 minutes, reaction time 5 minutes.

[0068] (a) Take 150μL reagent 1 and 2μL serum sample, mix well;

[0069] (b) Incubate the mixed solution at 37℃ for 5 minutes;

[0070] (c) Add 50μL reagent 2 again, incubate at 37℃ for 36s, record absorbance value A1, and read absorbance value A2 after 5min;

[0071] (d) Calculate the reaction degree = A2-A1.

[0072] The reaction degree (OD value) represents the degree of reaction between the beginning of the reaction and the end of the reaction in the immunonephelometry, and to some extent, it can reflect the sensitivity of the reaction. Table 2 below is the test calibration reaction degree result.

[0073] Table 2

[0074]

[0075] The calibration reaction degree fitting curve of each scheme is shown in Figure 1 .

[0076] Taking scheme 1 as the control group, the calibration curve shows obvious high value linearity, and the 200mg / L calibration reaction degree is lower than the 100mg / L reaction degree. With the addition of ionic liquid in each scheme, the reaction degree of each point decreases to some extent. Under the condition of the same anion in schemes 2, 3, 5, and 6, the chain length of the cation increases gradually, and the discrimination degree gradually increases compared with the control group. The longer the chain, the more obvious the reaction discrimination. Among them, the reaction of each point in schemes 5 and 6 can be seen to have obvious discrimination. Scheme 4 has a smaller anion radius than scheme 5 under the same cation condition, and the inhibition effect is better. The reaction degree of each point in scheme 4 has good discrimination compared with the control group. Schemes 4 and 7, and schemes 3 and 8 also show that under the same cation, the smaller the anion radius, the better the effect of ionic liquid on the system. Scheme 9 uses other types of ionic liquids, which has a certain inhibition effect, but the discrimination degree is not enough. Therefore, it is proved that the imidazole ring plays a key role in widening the linearity effect. In summary, the schemes with added ionic liquid have improved discrimination degree compared with the control group.

[0077] Linear detection: The high concentration retinol binding protein antigen was diluted with physiological saline to prepare a series of diluted samples with theoretical concentrations shown in the first column of the theoretical concentration in Table 1. Then, the reagents of each scheme were tested for linearity, and the results are shown in Table 3.

[0078] Table 3

[0079]

[0080] From the results in Table 3, the control group has no discrimination at high calibration values, and the linearity is poor when the concentration is higher than 100mg / L. Scheme 9 also fails to test at high calibration values, and the linear test also shows the same trend as the control group. Schemes 2, 3, 5, 7, and 8 have a certain degree of discrimination, and the relative deviation is controlled within 10%, which is acceptable. Scheme 6 has a certain degree of discrimination at high values, but the linear test result is poor at low values, and the relative deviation is more than 20%. Scheme 4 shows good linearity and discrimination, with a deviation of slightly more than 5% at 180mg / L, and the deviation at other concentrations is controlled within 5%. This test result is normal fluctuation in test results, and the linear test meets the requirements.

[0081] 2. The effect of microsphere size on linearity and determination of the optimal size range:

[0082] R1 reagent components are fixed, Tris-HCl buffer system, which consists of 50 mmol / L Tris-HCl buffer solution, pH 7.0, polyethylene glycol 6000 5 g / L, Tween 20 0.1%, EDTA-2Na 5 mmol / L, 1-hexyl-3-methyl imidazole chloride salt 75 g / L;

[0083] R2 scheme is: 0.5wt% goat anti-human retinol polyclonal antibody coated latex microspheres, 20mM Tris-HCl buffer solution, pH 7.5, ethylenediaminetetraacetic acid disodium 5mmol / L, wherein the particle size of the microspheres is changed as a single factor, and the scheme is shown in Table 4.

[0084] Table 4

[0085]

[0086] Detection steps:

[0087] Detection instrument: full-automatic biochemical analyzer (model: Biossays C8).

[0088] Detection temperature: 37℃.

[0089] Detection wavelength: main wavelength: 570nm, secondary wavelength: 800nm.

[0090] Reaction time: 10 minutes, of which incubation time 5 minutes, reaction time 5 minutes.

[0091] (a) Take 150μL reagent 1 and 2μL serum sample and mix well;

[0092] (b) Incubate the mixed solution at 37℃ for 5 minutes;

[0093] (c) Add 50μL reagent 2 again, incubate at 37℃ for 36s, record the absorbance value A1, and read the absorbance value A2 after 5min;

[0094] (d) Calculate the reaction degree = A2-A1.

[0095] The test calibration reaction degree results are shown in Table 5.

[0096] Table 5

[0097]

[0098]

[0099] The calibration reaction degree fitting curve of each scheme is as followsFigure 2 As shown.

[0100] The linearities under different particle sizes were tested, and the results are shown in Table 6 below.

[0101] Table 6

[0102]

[0103] Under different particle sizes of microspheres, 7.5% ionic liquid was added, and the sensitivity of low particle size microspheres was significantly reduced. In the linear test, 80 nm, 100 nm and 120 nm microspheres could meet the linear test in the test range of 0-200 mg / L. 80 nm and below had smaller particle size and more complex preparation process.

[0104] 3. Technical effect verification of adding ionic liquid:

[0105] The preferred ionic liquid, 1-hexyl-3-methylimidazole chloride, was used for verification, and the linearities of retinol binding protein and sample comparison were tested. The R1 scheme is shown in Table 7 below:

[0106] Table 7

[0107]

[0108] The R2 scheme is: 0.5wt% goat anti-human retinol polyclonal antibody coated latex microspheres with a particle size of 100 nm, pH 7.5 20mM Tris-HCl buffer, and 5mmol / L EDTA-2Na.

[0109] Detection steps:

[0110] Detection instrument: full-automatic biochemical analyzer (model: Biossays C8).

[0111] Detection temperature: 37°C.

[0112] Detection wavelength: main wavelength: 570nm, secondary wavelength: 800nm.

[0113] Reaction time: 10 minutes, including incubation time of 5 minutes and reaction time of 5 minutes.

[0114] (a) Mix 150μL of reagent 1 and 2μL of calibrator;

[0115] (b) Incubate the mixed solution at 37°C for 5 minutes;

[0116] (c) Add 50μL of reagent 2, incubate at 37°C for 36s, record the absorbance value A1, and read the absorbance value A2 after 5min;

[0117] (d) Calculate the reactivity = A2 - Al.

[0118] 3.1 Verification of preferred concentration of ionic liquid

[0119] Fixed R2 component, using physiological saline to dilute the calibration sample, test each point calibration reactivity, the results are shown in Table 8.

[0120] Table 8

[0121]

[0122] The curve fitting of each scheme is shown in Figure 3 .

[0123] From the reactivity and fitting curve results, with the increase of the concentration of ionic liquid, the inhibitory effect of reactivity is enhanced, which proves that the inhibition effect of immune response is also enhanced, so that each scheme and the control group have obvious distinction in the high value area. The concentration within 5%-10% can meet the linear test, and the concentration above 5% can make the reactivity between concentration points reach the maximum.

[0124] 3.2 Verification of sensitivity

[0125] Each scheme was tested for repeatability, and the sample concentration was selected based on the upper and lower limits of the reference range calculated by statistical calculation of the serum sample test results. The reference interval range is 22-53 mg / L. Sample 1 concentration: 20 mg / L, sample 2 concentration: 50 mg / L, each scheme was tested 10 times, and the test results are shown in Table 9.

[0126] Table 9

[0127]

[0128]

[0129] According to the test results, the repeatability coefficient of variation (CV) of each scheme test sample was calculated, the calculation formula was: CV = (SD / mean) * 100%, the results in the table showed that the sensitivity had certain influence on repeatability, and under the condition of sufficient sensitivity, each scheme test had good CV value.

[0130] 3.3 Linear interval test

[0131] Using physiological saline to dilute high concentration retinol binding protein antigen to prepare a series of dilution concentration samples, the theoretical concentration is shown in the first column of the theoretical concentration in Table 1, then each scheme reagent was tested for linearity, the results are shown in Table 10.

[0132] Table 10

[0133]

[0134] According to the linear test results in the table, after adding 5% and above ionic liquid, the above linear interval can basically meet the test. From the control group, the linearity can basically be measured to 100 mg / L, and both scheme 2 and scheme 3 can be measured to 200 mg / L, and the linear effect is better than that of the control group. Considering comprehensively, scheme 3 adds 7.5% ionic liquid, and compared with the control group, the linearity reaches the best standard.

[0135] 3.4 Verification of reducing non-specific binding:

[0136] Using scheme 3 and scheme 1 respectively, the samples on the market (Jiujiang Biological, item number: GSRRBP002) were compared and tested. The test results of the market manufacturers were taken as the x-axis, and scheme 1 and scheme 3 were taken as the y-axis. The absolute deviation = scheme test value - comparison manufacturer test value. The scatter plot was drawn, and the specific data is shown in Table 11.

[0137] Table 11

[0138]

[0139]

[0140]

[0141] The sample comparison fitting results are shown in Table 11. Figure 4

[0142] Because the control group lacks linearity in the calibration high value, the test results of samples higher than 100 mg / L are significantly decreased, the test correlation of samples below 100 mg / L is good, but the test difference between samples is large, and there is a large non-specific reaction. After adding ionic liquid, the calibration overall reaction decreases, and 50 samples are tested in the entire linear range and compared with the market product. The results show that the correlation is very high, y = 0.9877x + 0.7549 r = 0.9981, indicating that the non-specific reaction in each sample test is significantly reduced compared with the control group. 2

[0143] Example 2: Verification of anti-streptolysin O (ASO) project:

[0144] In the anti-streptolysin O, reagent 2 is the antibody liquid fixing component, which is composed of 0.25wt% latex microspheres coated with streptolysin antigen O (microsphere particle size 120nm), Tris-HCl 20mM and ethylenediaminetetraacetic acid disodium 5mmol / L at pH 7.5; the calibration sample is 1200IU / mL anti-streptolysin O, human serum matrix, which also includes preservatives, stabilizers, etc. The R1 reagent scheme is shown in Table 12.​​

[0145] Table 12

[0146]

[0147] Testing instrument: Fully automated biochemical analyzer (model: Biossays C8).

[0148] Detection temperature: 37℃.

[0149] Detection wavelength: Main wavelength: 600nm, Secondary wavelength: -.

[0150] Reaction time: 10 minutes, including 5 minutes of incubation time and 5 minutes of reaction time.

[0151] (a) Mix 150 μL of reagent 1 and 2.5 μL of serum sample.

[0152] (b) Incubate the mixed solution at 37°C for 5 minutes;

[0153] (c) Add another 75 μL of reagent 2, incubate at 37°C for at least 10 s, record the absorbance A1 at the measurement wavelength, and record the absorbance value A2 after another 300 s.

[0154] (d) Calculate the degree of reactivity = A2 - A1.

[0155] 1. Linear interval test:

[0156] The linearity test results for each embodiment are shown in Table 13 below.

[0157] Table 13

[0158]

[0159] The calibration response fitting curve is as follows Figure 5 As shown.

[0160] In the linearity test, the lack of ionic liquid added resulted in insufficient high-value calibration discrimination, leading to a large deviation in the high-value linearity test. In the scheme with 5% 1-hexyl-3-methylimidazolium chloride added, the linearity at each point was basically consistent with the theoretical value, and the linearity effect was better than the control group, fully meeting the linearity test results of 0-1200 IU / mL. As the concentration of ionic liquid added increased, the ratio of high-value detection results to low-value detection results was also generally higher than that of the control group, but the deviation of low values ​​increased. When the concentration was 5-7.5%, the deviation of low values ​​was controlled within 10%, but when the concentration was 10%, the deviation of low values ​​exceeded 10%.

[0161] 2. Validation to reduce nonspecific binding:

[0162] Selecting scheme 1: control group, scheme 2: 5% 1-hexyl-3-methylimidazolium chloride group, and the product of the listed manufacturer (Roche, item number: 04489403190) were tested for sample comparison. The test results of the listed manufacturer were taken as the x-axis, and the control group and the 5% 1-hexyl-3-methylimidazolium chloride group were taken as the y-axis. A scatter plot was drawn, and the sample comparison fitting results are shown in Figure 6

[0163] Figure 6 The comparison data show that after adding the ionic liquid, the correlation of the sample comparison has improved to some extent, from 0.9911 of scheme 1 to 0.9985. For the test samples (sample numbers 1-34) within the normal range, the absolute deviation after adding the ionic liquid is significantly reduced compared with the control group, and the absolute deviation in the medium and high value regions is also significantly improved, indicating that the test accuracy is improved, and a part of the non-specific reaction is reduced, so that the full segment test result is basically consistent with the comparison reagent result. Although the correlation of the control group is good, there is a large difference in the sample deviation, the low value region is seriously high, the medium and high value deviations are also obvious, and there is a large non-specific reaction. In summary of the above detection and comparison of the manufacturer's results, the addition of 5% 1-hexyl-3-methylimidazolium chloride in the ASO project can significantly improve the linearity effect and reduce part of the non-specific reaction to make the test result more accurate.

[0164] 3. Verification of sensitivity:

[0165] The repeatability verification test was performed for each scheme. The sample concentration was set based on the reference range obtained by statistical calculation of the serum sample detection result, and the reference interval range was ≤166 IU / mL. The sample 1 concentration was 50 IU / mL, and the sample 2 concentration was 150 IU / mL. Each scheme was tested 10 times, and the detection results are shown in Table 14.

[0166] Table 14

[0167]

[0168] The low value repeatability test of the control scheme and the addition of 5% ionic liquid was less than 2%. The low value repeatability was significantly improved with the increase of the ionic liquid concentration. When 7.5% ionic liquid was added, the corresponding cv was within 10%, and when 10% ionic liquid was added, the corresponding cv exceeded 10%. Scheme 4 had a large ionic liquid concentration, strong inhibition effect, and large deviation value in low value detection, resulting in poor sensitivity. However, on the basis of greatly improving the detection linear range of the scheme, the sacrifice of this degree of sensitivity is completely acceptable. The test results of scheme 2 and scheme 3 show that when detecting the ASO project, the non-specific reaction is reduced while the low value sensitivity is ensured.

[0169] ​Example 3: Validation of Ferritin project

[0170] Take Ferritin as an example, the components of the first reagent are: Tris-HCl buffer system, which includes 100 mM Tris-HCl buffer solution at pH 7.5, PEG 6000 5 g / L, EDTA-Na2 1 mM, Tween 20 0.1%, and ionic liquid 1-hexyl-3-methylimidazolium chloride, with the addition of concentrations of 0, 5%, 7.5%, and 10%, respectively. The components of the second reagent are: 0.1 wt% mouse anti-human ferritin polyclonal antibody coated latex microspheres (microsphere particle size 236 nm), 20 mM HEPES buffer solution at pH 6.5, and 0.9 g / L sodium azide.

[0171] Detection method:

[0172] Detection instrument: full-automatic biochemical analyzer (model: Biossays C8);

[0173] Detection temperature: 37°C;

[0174] Detection wavelength: main wavelength: 570 nm, secondary wavelength: -nm;

[0175] Reaction time: 10 minutes, including incubation time of 5 minutes and reaction time of 5 minutes.

[0176] (a) Mix 120 μL of reagent 1 and 8 μL of serum sample;

[0177] (b) Incubate the mixed solution at 37°C for 5 minutes;

[0178] (c) Add 40 μL of reagent 2, incubate at 37°C for 24 s, record the absorbance value A1, and then read the absorbance value A2 after 276 s; (d) Calculate ΔA = A2-A1.

[0179] The calibration data at different ionic liquid concentrations are shown in Table 15.

[0180] Table 15

[0181]

[0182] The fitting curve of the calibration reaction rate is shown in Figure 7 . The calibration data of different concentrations of ionic liquid show that the differentiation degree in the high value range is small when no ionic liquid is added, and the differentiation degree between each reaction rate increases continuously with the increase of the concentration of ionic liquid; when the ionic liquid is added to 10%, the low value sensitivity decreases due to the addition of excessive amount. The linear test results are shown in Table 16.

[0183] Table 16

[0184]

[0185] According to the above linear test results, when no ionic liquid is added, the relative deviation in the range of 500-1000 ng / mL is large, and when the ionic liquid is added, the accuracy of the test in the high value area gradually improves, so the ionic liquid addition concentration of 5%-10% also has the effect of improving the discrimination of the high value detection in the ferritin project. Considering the addition amount of 7.5% of 1-hexyl-3-methylimidazole chloride salt, the deviation of each point in the range of 500-1000 ng / mL is small, and the linear test effect is best.

[0186] Select scheme 1: control group, scheme 3: 5% 1-hexyl-3-methylimidazole chloride group, and the product of the listed manufacturer (Roche, item number 04885317190) are tested for sample comparison, the test results of the listed manufacturer are taken as the x-axis, and the control group and the 7.5% 1-hexyl-3-methylimidazole chloride group are taken as the y-axis, a scatter plot is drawn, the absolute deviation = scheme test value - comparison manufacturer test value, and the sample comparison fitting result is as shown in Figure 8 The specific test result data is shown in Table 17.

[0187] Table 17

[0188]

[0189]

[0190] From the comparison data in Table 17, after adding the ionic liquid, the correlation of the sample comparison is improved to a certain extent, from 0.971 of scheme 1 to 0.9889. When testing low value samples (sample numbers 1-15), the absolute deviation after adding the ionic liquid is significantly reduced, the absolute deviation in the medium and high value area is also significantly improved, a part of the non-specific reaction is reduced, the test accuracy is significantly improved, and the test results in the whole section are basically consistent with the comparison reagent results; the correlation of the control group is good, but there is a large difference in the sample deviation, the low value area is seriously high, the medium and high value deviation is also obvious, and there is a large non-specific reaction; in summary, the above detection and comparison manufacturer results show that the addition of 7.5% of 1-hexyl-3-methylimidazole chloride in the ferritin project can significantly improve the linear effect, reduce part of the non-specific reaction, and make the test results more accurate.

[0191] From the above description, it can be seen that the above embodiments of the present application achieve the following technical effects: by adding an ionic liquid in the latex detection, not only the linear range for detecting components such as retinol binding protein or anti-streptolysin O can be improved, but also the non-specific binding reaction in the detection can be reduced. By using the above detection reagent or kit, accurate detection of high concentration samples can be realized at a lower cost and with a simple method.

[0192] The above descriptions are only the preferred embodiments of the present application, not intended to limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall fall within the scope of the present application.

Claims

1. A latex immunoturbidimetric kit characterized in that, The kit comprises a first reagent, the first reagent comprising a buffer and an ionic liquid, The ionic liquid comprises an ionic liquid containing an imidazole structure, The ionic liquid containing an imidazole structure is 1-alkyl-3-methyl imidazole halide or 1-alkyl-3-methyl imidazole fluoride, The 1-alkyl-3-methyl imidazole halide is one or more of 1-ethyl-3-methyl imidazole bromide, 1-butyl-3-methyl imidazole bromide, 1-hexyl-3-methyl imidazole chloride or 1-hexyl-3-methyl imidazole bromide; The 1-alkyl-3-methyl imidazole fluoride is 1-alkyl-3-methyl imidazole tetrafluoroborate; The 1-alkyl-3-methyl imidazole tetrafluoroborate comprises 1-butyl-3-methyl imidazole tetrafluoroborate or 1-hexyl-3-methyl imidazole tetrafluoroborate; The mass content of the 1-hexyl-3-methyl imidazole chloride in the first reagent is 5%-10%; The mass content of 1-ethyl-3-methyl imidazole bromide, 1-butyl-3-methyl imidazole bromide, 1-hexyl-3-methyl imidazole bromide, 1-butyl-3-methyl imidazole tetrafluoroborate or 1-hexyl-3-methyl imidazole tetrafluoroborate in the first reagent is 5%; The kit further comprises a second reagent, the second reagent comprising an antibody or an antigen-coated latex microsphere; The antibody comprises a retinol antibody or a ferritin antibody, and the antigen comprises a streptolysin O antigen, In the kit containing the retinol antibody, the particle size of the latex microsphere is 80-180 nm; In the kit containing the streptolysin O antigen, the particle size of the latex microsphere is 100-250 nm; In the kit containing the ferritin antibody, the particle size of the latex microsphere is 200-300 nm.

2. The kit of claim 1, wherein The buffer comprises any one of Tris-HCl buffer, Na2HPO4-NaH2PO4 buffer, disodium hydrogen phosphate-citric acid buffer, glyoxal-HCl buffer and barbiturate buffer.

3. The kit of claim 2, wherein The pH value of the buffer is 5.0-10.

0.

4. The kit of claim 3, wherein The buffer comprises the Tris-HCl buffer with a pH value of 7.0-9.0 and a concentration of 50-500 mmol / L.

5. The kit of claim 2, wherein The buffer further comprises one or more of inorganic salt, polyethylene glycol, ion chelating agent or surfactant.

6. The kit of claim 5, wherein The inorganic salt comprises one or more of sodium chloride, potassium chloride, sodium acetate or magnesium chloride.

7. The kit of claim 6, wherein The concentration of the inorganic salt is 5-10 g / L.

8. The kit of claim 5, wherein The ion chelating agent is any one of EDTA-2Na, EDTA-4Na, potassium sodium tartrate and ammonium citrate.

9. The kit of claim 5, wherein The polyethylene glycol comprises one or more of PEG800, PEG2000, PEG6000, PEG8000, PEG10000 or PEG20000.

10. The kit of claim 5, wherein The surfactant comprises one or more of Tween 20, Tween 80, Tritin X100, Brij 35, Brij 58 or Span 80.

11. The kit of any one of claims 1-10, wherein, In the kit, the first reagent and the second reagent are independently arranged.

12. The kit of any one of claims 1-10, wherein, The kit also includes a calibrator including the corresponding antigen or antibody, and a human serum matrix, a preservative, and a stabilizer.

13. The kit of claim 12, wherein The preservative includes one or more of sodium azide, ProClin 300, ProClin 150, ProClin 950, gentamicin, penicillin, BiT, potassium sorbate, or sodium benzoate.

14. The kit of claim 12, wherein The stabilizer includes one or more of bovine serum albumin, trehalose, mannitol, ethylene glycol, glycerol, or casein.

15. The kit of claim 1, wherein In the kit containing the retinol antibody, the particle size of the latex microspheres is 80-120 nm.

16. The kit of claim 1, wherein In the kit containing the streptolysin O antigen, the particle size of the latex microspheres is 100-150 nm.

17. The kit of claim 1, wherein In the kit containing the ferritin antibody, the particle size of the latex microspheres is 220-250 nm.

18. The kit of any one of claims 1-10, wherein, The latex microspheres are of the same size.

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