A latex enhanced immunoturbidimetric detection reagent and its application

Abstract

The present invention discloses a latex-enhanced immunoturbidimetric detection reagent, which relates to the technical field of medical detection and is prepared from the following components: inorganic salts, chelating agents, coagulants, excipients, nonionic surfactants, antibiotics, preservatives, antibody stabilizers, suspending agents, latex microspheres labeled with antibodies, high-purity liquid paraffin, zwitterionic organic salts, amino-terminated water-soluble hyperbranched polyamides, 1,3-bis((tris(hydroxymethyl)methyl)amino)propane, hydroxyethyl hexahydro-1,3,5-triazine, and buffer solutions. This latex-enhanced immunoturbidimetric detection reagent has good stability, high repeatability, excellent sensitivity, and a long shelf life.

Description

Technical Field

[0001] The present invention relates to the technical field of medical detection, and particularly relates to a latex-enhanced immunoturbidimetric detection reagent and its application. Background Art

[0002] The latex-enhanced immunoturbidimetric assay reagent is a new type of immunodiagnostic reagent that uses specific antibodies to coat latex particles and bind to antigens in the sample. By detecting the turbidity of the reaction solution, quantitative analysis of the sample is achieved. It has the advantages of high detection sensitivity, strong specificity, fast detection speed, simple operation, low cost, etc., and can be used for large-scale detection on various biochemical analyzers. It is widely used in the clinical diagnosis of diseases such as cardiovascular and cerebrovascular diseases, tumors, kidney diseases, liver diseases, and diabetes, and has become one of the mainstream methods for clinical serum protein quantitative detection.

[0003] Currently, commercially available latex-enhanced immunoturbidimetric detection reagents and kits are all double reagents, consisting of reaction buffer 1 (R1) and antibody-labeled latex-enhanced particle reagent 2 (R2). Premixing R1 and R2 easily causes inactivation of the antibodies in R2 and aggregation of latex particles, and the kit cannot be stored for a long time. Especially for the determination of whole blood samples, currently commercially available kits require two steps of hemolysis first and then determination, which is time-consuming and has poor repeatability.

[0004] To solve the above problems, the Chinese invention patent with the authorization announcement number CN104049085B discloses a CRP latex-enhanced immunoturbidimetric assay reagent. The CRP latex-enhanced immunoturbidimetric assay reagent includes CRP antibody-labeled latex particles, buffer, surfactant, inorganic salt, stabilizer, suspending agent, excipient, and preservative. The invention also provides a single-reagent CRP latex-enhanced immunoturbidimetric assay kit and its application. The reagent of the invention has a simple composition, is simple and convenient to operate, has a fast reaction, good reagent stability, high sensitivity, good repeatability, simple instrument used, wide adaptability to instruments, and low cost, and can be widely used in large, medium, and small hospitals and outpatient clinics for rapid clinical diagnosis of infections and evaluation of infection prognosis. However, it only discloses that it is applicable to the CRP project, and there is still a lack of a general-purpose latex-enhanced immunoturbidimetric detection reagent applicable to multiple projects (including CRP) on the market. Moreover, there is still room for improvement in the repeatability, sensitivity, stability, and shelf life of this reagent.

[0005] It can be seen that developing a single-reagent type latex-enhanced immunoturbidimetric detection reagent with good stability, high repeatability, good sensitivity, and long shelf life and its application meets the market demand, has broad market value and application prospects, and is of great significance for promoting the development of the latex-enhanced immunoturbidimetric detection field. Summary of the Invention

[0006] The object of the present invention is to provide a single-reagent latex enhanced immunoturbidimetric detection reagent with good stability, high repeatability, excellent sensitivity and long shelf life, as well as its application, in order to overcome the deficiencies of the prior art.

[0007] To achieve the above object, the technical solution adopted by the present invention is: a latex enhanced immunoturbidimetric detection reagent, which is prepared from the following components by weight percentage: inorganic salts 0.5 - 2 wt%, chelating agent 0.1 - 1 wt%, coagulant 0.4 - 2.2 wt%, excipient 0.9 - 5.2 wt%, non-ionic surfactant 0.5 - 1 wt%, antibiotic 0.1 - 0.3 wt%, preservative 0.04 - 0.6 wt%, antibody stabilizer 0.4 - 2.2 wt%, suspending agent 0.9 - 5 wt%, latex microspheres labeled with antibodies 0.1 - 0.2 wt%, high-purity liquid paraffin 0.1 - 0.6 wt%, zwitterionic organic salt 0.1 - 0.3 wt%, amino-terminated water-soluble hyperbranched polyamide 0.01 - 0.12 wt%, 1,3-bis((tris(hydroxymethyl)methylamino)propane 0.03 - 0.1 wt%, hydroxyethyl hexahydro-s-triazine 0.01 - 0.06 wt%, and the balance is buffer solution.

[0008] Preferably, the inorganic salt is one or a combination of several of sodium chloride, potassium chloride, sodium sulfate, and potassium sulfate.

[0009] Preferably, the chelating agent is ethylenediaminetetraacetic acid.

[0010] Preferably, the coagulant is at least one of PEG6000 and dextran.

[0011] Preferably, the excipient is at least one of mannitol, glucose, sucrose, and maltose.

[0012] Preferably, the non-ionic surfactant is at least one of Triton X-100 and Tween 40.

[0013] Preferably, the antibiotic is gentamicin; the preservative is at least one of sodium azide and ProClin300; the antibody stabilizer is at least one of bovine serum albumin and casein.

[0014] Preferably, the suspending agent is at least one of ethylene glycol and glycerol.

[0015] Preferably, the latex microspheres labeled with antibodies are latex microspheres labeled with antibodies against the target analyte, and can be prepared by conventional methods in the art.

[0016] Preferably, the latex microspheres are at least one of polystyrene latex microspheres with carboxyl groups on the surface or polystyrene latex microspheres with amino groups on the surface.

[0017] Preferably, the latex microspheres include at least two different sizes of latex microspheres with an average particle size of 60 - 150 nm and 300 - 450 nm.

[0018] Preferably, the surface carboxyl density of the carboxyl group-containing polystyrene latex microspheres is 0.05 - 0.35 mmol / g, and the surface amino density of the amino group-containing polystyrene latex microspheres is 0.1 - 0.2 mmol / g.

[0019] Preferably, the high-purity liquid paraffin is white oil No. 26 with a purity of ≥99.9%, provided by Jingmen Petrochemical.

[0020] Preferably, the zwitterionic organic salt is at least one of pyridinium hydroxypropanesulfonate and 1-benzylpyridinium-3-carboxylate.

[0021] Preferably, the amino-terminated water-soluble hyperbranched polyamide is prepared by the method of Example 1 of the Chinese invention patent with the authorization announcement number CN1232567C.

[0022] Preferably, the buffer solution is any one of 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid-sodium hydroxide buffer solution (DIPSO-NaOH), 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid-sodium hydroxide buffer solution (HEPPS-NaOH), 3-(N-morpholino)propanesulfonic acid-sodium hydroxide buffer solution (MOPS-NaOH), N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid-NaOH buffer solution (HEPES-NaOH), tris(hydroxymethyl)aminomethane-HCl buffer solution (Tris-HCl), phosphate buffer solution, glycine-sodium hydroxide buffer solution, and barbital buffer solution. The buffering capacity of the buffer solution is required to be in the pH range of 7.0 - 9.0 and the concentration range is 10 - 100 mmol / L.

[0023] Another object of the present invention is to provide a preparation method of the latex-enhanced immunoturbidimetric detection reagent, including the following steps: mixing the components of the single-reagent type latex-enhanced immunoturbidimetric detection reagent evenly by weight percentage to obtain the latex-enhanced immunoturbidimetric detection reagent.

[0024] Another object of the present invention is to provide an application of the latex-enhanced immunoturbidimetric detection reagent in a single-reagent CRP latex-enhanced immunoturbidimetric assay kit.

[0025] Due to the application of the above technical solutions, the present invention has the following beneficial effects:

[0026] (1) The preparation method of the latex enhanced immunoturbidimetric detection reagent disclosed by the present invention has a simple process, convenient operation control, high preparation efficiency and high qualified product rate, is suitable for large-scale production, and has high popularization and application value.

[0027] (2) The latex enhanced immunoturbidimetric detection reagent disclosed by the present invention is prepared from the following components by weight percentage: inorganic salt 0.5 - 2 wt%, chelating agent 0.1 - 1 wt%, coagulant 0.4 - 2.2 wt%, excipient 0.9 - 5.2 wt%, nonionic surfactant 0.5 - 1 wt%, antibiotic 0.1 - 0.3 wt%, preservative 0.04 - 0.6 wt%, antibody stabilizer 0.4 - 2.2 wt%, suspending agent 0.9 - 5 wt%, latex microspheres labeled with antibody 0.1 - 0.2 wt%, high-purity liquid paraffin 0.1 - 0.6 wt%, zwitterionic organic salt 0.1 - 0.3 wt%, terminal amino water-soluble hyperbranched polyamide 0.01 - 0.12 wt%, 1,3-bis((tris(hydroxymethyl)methylamino)propane 0.03 - 0.1 wt%, hydroxyethyl hexahydro-s-triazine 0.01 - 0.06 wt%, and the balance is buffer solution. Through the mutual cooperation and joint action of each component, the prepared latex enhanced immunoturbidimetric detection reagent has good stability, high repeatability, good sensitivity and long shelf life.

[0028] (3) The latex enhanced immunoturbidimetric detection reagent disclosed by the present invention adopts a single reagent, which is more convenient to operate than the traditional double reagent, and can effectively avoid antibody inactivation and latex particle aggregation; the latex microspheres include at least two different sizes of latex microspheres with an average particle size of 60 - 150 nm and 300 - 450 nm, which increases the turbidity during antibody reaction, thereby increasing the sensitivity of the test reaction, and at the same time can ensure the linearity of the reagent. The addition of terminal amino water-soluble hyperbranched polyamide, 1,3-bis((tris(hydroxymethyl)methylamino)propane, hydroxyethyl hexahydro-s-triazine, and zwitterionic organic salt, and their interaction with other raw material components can bind to the antibody through weak interaction methods such as hydrogen bonds. On the one hand, it inhibits the latex aggregation phenomenon caused by the binding of other non-specific proteins or small molecules to the antibody-sensitized latex, and on the other hand, it does not affect the specific binding of the antibody to the antigen. Thereby improving the stability and detection accuracy of the reagent and improving the detection range.

[0029] (4) For the latex enhanced immunoturbidimetric detection reagent disclosed by the present invention, the addition of high-purity liquid paraffin can prevent liquid volatilization, and cooperate with the zwitterionic organic salt to effectively improve the stability of the reagent. Specific embodiments

[0030] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are only examples, and those skilled in the art can think of other obvious variations.

[0031] Example 1

[0032] A latex enhanced immunoturbidimetric detection reagent is prepared from the following components by weight percentage: inorganic salt 0.5 wt%, chelating agent 0.1 wt%, coagulant 0.4 wt%, excipient 0.9 wt%, nonionic surfactant 0.5 wt%, antibiotic 0.1 wt%, preservative 0.04 wt%, antibody stabilizer 0.4 wt%, suspending agent 0.9 wt%, latex microspheres labeled with antibody 0.1 wt%, high-purity liquid paraffin 0.1 wt%, zwitterionic organic salt 0.1 wt%, amino-terminated water-soluble hyperbranched polyamide 0.01 wt%, 1,3-bis((tris(hydroxymethyl)methylamino)propane 0.03 wt%, hydroxyethyl hexahydro-s-triazine 0.01 wt%, and the balance is buffer solution.

[0033] The inorganic salt is sodium chloride; the chelating agent is ethylenediaminetetraacetic acid; the coagulant is PEG6000; the excipient is mannitol; the nonionic surfactant is Triton X-100; the antibiotic is gentamicin; the preservative is sodium azide; the antibody stabilizer is bovine serum albumin; the suspending agent is ethylene glycol; the latex microspheres labeled with antibody are latex microspheres labeled with anti-target analyte antibody, prepared by the conventional method in the art (refer to the method in CN104049085); the latex microspheres are composed of polystyrene latex microspheres with carboxyl groups on the surface and polystyrene latex microspheres with amino groups on the surface mixed in a mass ratio of 1:1; the average particle size of the polystyrene latex microspheres with carboxyl groups on the surface is 60 nm, and the average particle size of the polystyrene latex microspheres with amino groups on the surface is 450 nm; the surface carboxyl density of the polystyrene latex microspheres with carboxyl groups on the surface is 0.05 mmol / g, and the surface amino density of the polystyrene latex microspheres with amino groups on the surface is 0.1 mmol / g.

[0034] The high-purity liquid paraffin is No. 26 white oil with a purity ≥ 99.9%, provided by Jingmen Petrochemical; the zwitterionic organic salt is pyridinium hydroxypropyl sulfonate; the amino-terminated water-soluble hyperbranched polyamide is prepared by the method of Example 1 of the Chinese invention patent with the authorization announcement number CN1232567C.

[0035] The buffer solution is 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid-sodium hydroxide buffer solution (DIPSO-NaOH). The buffering capacity of the buffer solution is required to be in the pH range of 7.0 - 9.0 and the concentration range is 10 mmol / L.

[0036] A preparation method of the latex enhanced immunoturbidimetric detection reagent comprises the following steps: Mixing each component of the single-reagent type latex enhanced immunoturbidimetric detection reagent evenly by weight percentage to obtain the latex enhanced immunoturbidimetric detection reagent.

[0037] Example 2

[0038] A latex enhanced immunoturbidimetric detection reagent is prepared from the following components by weight percentage: inorganic salt 0.9 wt%, chelating agent 0.3 wt%, coagulant 1.2 wt%, excipient 2.2 wt%, non-ionic surfactant 0.6 wt%, antibiotic 0.15 wt%, preservative 0.15 wt%, antibody stabilizer 0.9 wt%, suspending agent 2 wt%, latex microspheres labeled with antibody 0.13 wt%, high-purity liquid paraffin 0.2 wt%, zwitterionic organic salt 0.15 wt%, amino-terminated water-soluble hyperbranched polyamide 0.05 wt%, 1,3-bis((tris(hydroxymethyl)methylamino)propane 0.06 wt%, hydroxyethyl hexahydro-s-triazine 0.02 wt%, and the balance is buffer solution.

[0039] The inorganic salt is potassium chloride; the chelating agent is ethylenediaminetetraacetic acid; the coagulant is dextran; the excipient is glucose; the non-ionic surfactant is Tween 40; the antibiotic is gentamicin; the preservative is ProClin300; the antibody stabilizer is casein; the suspending agent is ethylene glycol; the latex microspheres labeled with antibody are latex microspheres labeled with anti-target analyte antibody, prepared by the conventional method in the art (refer to the method in CN104049085); the latex microspheres are composed of polystyrene latex microspheres with carboxyl groups on the surface and polystyrene latex microspheres with amino groups on the surface mixed in a mass ratio of 2:3; the average particle size of the polystyrene latex microspheres with carboxyl groups on the surface is 100 nm, and the average particle size of the polystyrene latex microspheres with amino groups on the surface is 420 nm; the carboxyl density on the surface of the polystyrene latex microspheres with carboxyl groups on the surface is 0.15 mmol / g, and the amino density on the surface of the polystyrene latex microspheres with amino groups on the surface is 0.13 mmol / g.

[0040] The high-purity liquid paraffin is No. 26 white oil with a purity ≥ 99.9%, provided by Jingmen Petrochemical; the zwitterionic organic salt is 1-benzylpyridinium-3-carboxylate; the amino-terminated water-soluble hyperbranched polyamide is prepared by the method of Example 1 of the Chinese invention patent with the authorization announcement number CN1232567C; the buffer solution is 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid-sodium hydroxide buffer solution (HEPPS-NaOH), and the buffering capacity requirement of the buffer solution is in the pH range of 7.0 - 9.0 and the concentration range is 30 mmol / L.

[0041] A preparation method of the latex enhanced immunoturbidimetric detection reagent comprises the following steps: Mixing each component of the single-reagent type latex enhanced immunoturbidimetric detection reagent evenly by weight percentage to obtain the latex enhanced immunoturbidimetric detection reagent.

[0042] Example 3

[0043] A latex enhanced immunoturbidimetric detection reagent is prepared from the following components by weight percentage: inorganic salts 1.3 wt%, chelating agent 0.6 wt%, coagulant 1.4 wt%, excipient 3.2 wt%, non-ionic surfactant 0.8 wt%, antibiotic 0.2 wt%, preservative 0.35 wt%, antibody stabilizer 1.4 wt%, suspending agent 3.5 wt%, latex microspheres labeled with antibody 0.15 wt%, high-purity liquid paraffin 0.4 wt%, zwitterionic organic salt 0.2 wt%, amino-terminated water-soluble hyperbranched polyamide 0.08 wt%, 1,3-bis((tris(hydroxymethyl)methylamino)propane 0.07 wt%, hydroxyethyl hexahydro-s-triazine 0.035 wt%, and the balance is buffer solution.

[0044] The inorganic salts are a mixture of sodium chloride, potassium chloride, sodium sulfate, and potassium sulfate in a mass ratio of 1:2:1:2; the chelating agent is ethylenediaminetetraacetic acid; the coagulant is a mixture of PEG6000 and dextran in a mass ratio of 1:3; the excipient is a mixture of mannitol, glucose, sucrose, and maltose in a mass ratio of 1:2:2:3; the non-ionic surfactant is a mixture of Triton X-100 and Tween 40 in a mass ratio of 3:5; the antibiotic is gentamicin; the preservative is a mixture of sodium azide and ProClin300 in a mass ratio of 2:3; the antibody stabilizer is a mixture of bovine serum albumin and casein in a mass ratio of 3:1; the suspending agent is a mixture of ethylene glycol and glycerol in a mass ratio of 2:3.

[0045] The latex microspheres labeled with antibody are latex microspheres labeled with an antibody against the target analyte, and are prepared by a conventional method in the art (refer to the method in CN104049085); the latex microspheres are a mixture of polystyrene latex microspheres with carboxyl groups on the surface and polystyrene latex microspheres with amino groups on the surface in a mass ratio of 1:3; the average particle size of the polystyrene latex microspheres with carboxyl groups on the surface is 130 nm; the average particle size of the polystyrene latex microspheres with amino groups on the surface is 350 nm; the surface carboxyl density of the polystyrene latex microspheres with carboxyl groups on the surface is 0.3 mmol / g, and the surface amino density of the polystyrene latex microspheres with amino groups on the surface is 0.18 mmol / g.

[0046] The high-purity liquid paraffin is white oil No. 26 with a purity of ≥99.9%, provided by Jingmen Petrochemical; the amphoteric organic ion salt is a mixture of pyridinium hydroxypropanesulfonate and 1-benzylpyridinium-3-carboxylate in a mass ratio of 1:1; the amino-terminated water-soluble hyperbranched polyamide is prepared according to the method of Example 1 of the Chinese invention patent with the authorization announcement number CN1232567C; the buffer solution is 3-(N-morpholino)propanesulfonic acid-sodium hydroxide buffer solution (MOPS-NaOH), and the buffering capacity of the buffer solution is required to be in the pH range of 7.0-9.0 and the concentration range is 60 mmol / L.

[0047] A preparation method of the latex-enhanced immunoturbidimetric detection reagent comprises the following steps: mixing each component of the single-reagent type latex-enhanced immunoturbidimetric detection reagent evenly by weight percentage to obtain the latex-enhanced immunoturbidimetric detection reagent.

[0048] Example 4

[0049] A latex-enhanced immunoturbidimetric detection reagent is prepared from the following components by weight percentage: inorganic salt 1.8 wt%, chelating agent 0.9 wt%, coagulant 2 wt%, excipient 5 wt%, non-ionic surfactant 0.9 wt%, antibiotic 0.25 wt%, preservative 0.5 wt%, antibody stabilizer 2 wt%, suspending agent 4.5 wt%, latex microspheres labeled with antibody 0.18 wt%, high-purity liquid paraffin 0.5 wt%, amphoteric organic ion salt 0.25 wt%, amino-terminated water-soluble hyperbranched polyamide 0.1 wt%, 1,3-bis((tris(hydroxymethyl)methylamino)propane 0.09 wt%, hydroxyethyl hexahydro-s-triazine 0.05 wt%, and the balance is buffer solution.

[0050] The inorganic salt is potassium sulfate; the chelating agent is ethylenediaminetetraacetic acid; the coagulant is PEG6000; the excipient is maltose; the non-ionic surfactant is Triton X-100; the antibiotic is gentamicin; the preservative is sodium azide; the antibody stabilizer is bovine serum albumin; the suspending agent is glycerol; the latex microspheres labeled with antibody are latex microspheres labeled with anti-target analyte antibody, prepared by the conventional method in the art (refer to the method in CN104049085).

[0051] The latex microspheres are composed of a mixture of polystyrene latex microspheres with carboxyl groups on the surface and polystyrene latex microspheres with amino groups on the surface at a mass ratio of 2:1; the average particle size of the polystyrene latex microspheres with carboxyl groups on the surface is 140 nm; the average particle size of the polystyrene latex microspheres with amino groups on the surface is 330 nm; the surface carboxyl density of the polystyrene latex microspheres with carboxyl groups on the surface is 0.33 mmol / g, and the surface amino density of the polystyrene latex microspheres with amino groups on the surface is 0.18 mmol / g.

[0052] The high-purity liquid paraffin is No. 26 white oil with a purity ≥ 99.9%, provided by Jingmen Petrochemical; the zwitterionic organic salt is pyridinium hydroxypropane sulfonate; the amino-terminated water-soluble hyperbranched polyamide is prepared by the method of Example 1 of the Chinese invention patent with the authorization announcement number CN1232567C; the buffer solution is N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid-NaOH buffer solution (HEPES-NaOH), and the buffering capacity of the buffer solution is required to be in the pH range of 7.0 - 9.0 and the concentration range is 90 mmol / L.

[0053] A preparation method of the latex enhanced immunoturbidimetric detection reagent includes the following steps: Mix each component of the single-reagent type latex enhanced immunoturbidimetric detection reagent evenly by weight percentage to obtain the latex enhanced immunoturbidimetric detection reagent.

[0054] Example 5

[0055] A latex enhanced immunoturbidimetric detection reagent is prepared from the following components by weight percentage: inorganic salts 2 wt%, chelating agent 1 wt%, coagulant 2.2 wt%, excipient 5.2 wt%, non-ionic surfactant 1 wt%, antibiotic 0.3 wt%, preservative 0.6 wt%, antibody stabilizer 2.2 wt%, suspending agent 5 wt%, latex microspheres labeled with antibody 0.2 wt%, high-purity liquid paraffin 0.6 wt%, zwitterionic organic salt 0.3 wt%, amino-terminated water-soluble hyperbranched polyamide 0.12 wt%, 1,3-bis((tris(hydroxymethyl)methylamino)propane 0.1 wt%, hydroxyethyl hexahydro-s-triazine 0.06 wt%, and the balance is buffer solution.

[0056] The inorganic salt is sodium sulfate; the chelating agent is ethylenediaminetetraacetic acid; the coagulant is PEG6000; the excipient is sucrose; the non-ionic surfactant is Triton X-100; the antibiotic is gentamicin; the preservative is sodium azide; the antibody stabilizer is casein; the suspending agent is glycerol; the latex microspheres labeled with antibodies are latex microspheres labeled with antibodies against the target analyte, prepared by conventional methods in the art (refer to the method in CN104049085); the latex microspheres are composed of polystyrene latex microspheres with carboxyl groups on the surface and polystyrene latex microspheres with amino groups on the surface mixed in a mass ratio of 1:2; the average particle size of the polystyrene latex microspheres with carboxyl groups on the surface is 150 nm; the average particle size of the polystyrene latex microspheres with amino groups on the surface is 300 nm; the carboxyl density on the surface of the polystyrene latex microspheres with carboxyl groups on the surface is 0.35 mmol / g, and the amino density on the surface of the polystyrene latex microspheres with amino groups on the surface is 0.2 mmol / g.

[0057] The high-purity liquid paraffin is white oil No. 26 with a purity of ≥99.9%, provided by Jingmen Petrochemical; the zwitterionic organic salt is pyridinium hydroxypropylsulfonate; the water-soluble hyperbranched polyamide with terminal amino groups is prepared according to the method of Example 1 of the Chinese invention patent with the authorization announcement number CN1232567C; the buffer solution is tris(hydroxymethyl)aminomethane-HCl buffer solution (Tris-HCl), and the adjustment ability of the buffer solution requires a pH range of 7.0 - 9.0 and a concentration range of 100 mmol / L.

[0058] A preparation method of the latex enhanced immunoturbidimetric detection reagent comprises the following steps: mixing each component of the single-reagent type latex enhanced immunoturbidimetric detection reagent according to the weight percentage, and then the latex enhanced immunoturbidimetric detection reagent is obtained.

[0059] Comparative Example 1

[0060] A latex enhanced immunoturbidimetric detection reagent and its preparation method are basically the same as those in Example 1, except that high-purity liquid paraffin and zwitterionic organic salt are not added.

[0061] Comparative Example 2

[0062] A latex enhanced immunoturbidimetric detection reagent and its preparation method are basically the same as those in Example 1, except that water-soluble hyperbranched polyamide with terminal amino groups, 1,3-bis((tris(hydroxymethyl)methylamino)propane) and hydroxyethyl hexahydro-s-triazine are not added.

[0063] To further illustrate the beneficial technical effects of the latex-enhanced immunoturbidimetric detection reagents involved in the embodiments of the present invention, relevant performance tests were carried out on the latex-enhanced immunoturbidimetric detection reagents involved in Examples 1-5 and Comparative Examples 1-2. The test methods are as follows: The reagents prepared in each of the above examples were tested with a Beckman AU480 automatic biochemical analyzer. The test wavelength was 600 nm. 4 μL of CRP calibration products (dissolving commercially available CRP pure products in 50 mM Tris-HCl, pH 7.5, 0.85% NaCl, 0.5% BSA, 0.1% sodium azide, and the CRP concentrations were 0 mg / L, 5 mg / L, 15 mg / L, 50 mg / L, 100 mg / L, 150 mg / L respectively) were taken, and then 300 μL of the reagents prepared in each example were added. After incubation at 37°C for 3 min, the absorbance A1 was read after 20 s, and the absorbance A2 was read after incubation at 37°C for 2 min 30 s. Then the reaction absorbance ΔA = A2 - A1; First, multi-point calibration was performed using standard products, and a spline function was used for calculation to obtain a calibration curve. The PCT concentration in the sample can be obtained by comparing the absorbance change of the sample with the standard curve. The above examples were verified for analytical sensitivity, accuracy, precision, and stability, etc.

[0064] Table 1

[0065] Project Detection range Accuracy (recovery rate) Precision (CV) Stability Unit ng / mL ％ ％ Month Example 1 0.01-27.5 99.73 0.97 24 Example 2 0.01-27.7 99.79 0.92 24 Example 3 0.01-27.8 99.83 0.90 24 Example 4 0.01-27.8 99.88 0.86 24 Example 5 0.01-28.0 99.91 0.83 24 Comparative Example 1 0.2-28.2 98.67 1.32 18 Comparative Example 2 0.2-28.0 98.45 1.38 18

[0066] As can be seen from Table 1, the products of the embodiments of the present invention have more excellent sensitivity, accuracy, precision, and stability than the comparative examples. The addition of high-purity liquid paraffin, zwitterionic organic salts, amino-terminated water-soluble hyperbranched polyamide, 1,3-bis((tris(hydroxymethyl)methylamino)propane), and hydroxyethyl hexahydro-s-triazine is beneficial to improving the above performances.

[0067] The above embodiments are only used to illustrate the technical concept and characteristics of the present invention, and the purpose is to enable those who are familiar with this technology to understand the content of the present invention and implement it accordingly. It cannot be used to limit the protection scope of the present invention. All equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. A latex enhanced immunoturbidimetric detection reagent, characterized in that: The invention comprises the following components in percentage by weight: 0.5-2wt% of inorganic salt, 0.1-1wt% of chelating agent, 0.4-2.2wt% of coagulant, 0.9-5.2wt% of excipient, 0.5-1wt% of nonionic surfactant, 0.1-0.3wt% of antibiotic, 0.04-0.6wt% of preservative, 0.4-2.2wt% of antibody stabilizer, 0.9-5wt% of suspending agent, 0.1-0.2wt% of latex microspheres labeled with antibody, 0.01-0.2wt% of high-purity liquid paraffin, 0.01-0.2wt% of slurry, 0.02-0.08wt% of slurry, 0.01-0.08 ... .1-0.6wt%, amphoteric organic ion salt 0.1-0.3wt%, terminal amino water-soluble hyperbranched polyamide 0.01-0.12wt%, 1,3-bis((trihydroxymethyl)methylamino)propane 0.03-0.1wt%, hydroxyethylhexahydro-s-triazine 0.01-0.06wt%, and the remainder is buffer solution; the high-purity liquid paraffin is No. 26 white oil with a purity of ≥99.9%; the amphoteric organic ion salt is at least one of hydroxypropane sulfonate pyridinium salt and 1-benzylpyridine-3-carboxylate.

2. The latex-enhanced immunoturbidimetric detection reagent according to claim 1, characterized in that: The inorganic salt is one or a combination of sodium chloride, potassium chloride, sodium sulfate, potassium sulfate; and the chelating agent is ethylenediaminetetraacetic acid.

3. The latex-enhanced immunoturbidimetric detection reagent according to claim 1, characterized in that: The coagulant is at least one of PEG6000 and dextran; the excipient is at least one of mannitol, glucose, sucrose and maltose.

4. The latex-enhanced immunoturbidimetric detection reagent according to claim 1, characterized in that: The nonionic surfactant is at least one of Triton X-100 and Tween 40; and the antibiotic is gentamicin.

5. The latex-enhanced immunoturbidimetric detection reagent according to claim 1, characterized in that: The preservative is at least one of sodium azide and ProClin300; the antibody stabilizer is at least one of bovine serum albumin and casein; and the suspending agent is at least one of ethylene glycol and glycerol.

6. The latex-enhanced immunoturbidimetric detection reagent according to claim 1, characterized in that: The antibody-labeled latex microspheres are latex microspheres labeled with anti-target detection antibody; the latex microspheres are at least one of polystyrene latex microspheres containing carboxyl groups on the surface or polystyrene latex microspheres containing amino groups on the surface; the latex microspheres include at least two different sizes of latex microspheres with average particle sizes of 60-150nm and 300-450nm; the surface carboxyl density of the polystyrene latex microspheres containing carboxyl groups on the surface is 0.05-0.35mmol / g, and the surface amino density of the polystyrene latex microspheres containing amino groups on the surface is 0.1-0.2mmol / g.

7. The latex-enhanced immunoturbidimetric detection reagent according to claim 1, characterized in that: The buffer is any one of 3-[N,N-di(hydroxyethyl)amino]-2-hydroxypropanesulfonic acid-sodium hydroxide buffer (DIPSO-NaOH), 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid-sodium hydroxide buffer (HEPPS-NaOH), 3-(N-morpholino)propanesulfonic acid-sodium hydroxide buffer (MOPS-NaOH), N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid-NaOH buffer (HEPES-NaOH), tris(hydroxymethylaminomethane)-HCl buffer (Tris-HCl), phosphate buffer, glycine-NaOH buffer, and barbiturate buffer. The buffer has an adjustment capability of a pH range of 7.0-9.0 and a concentration range of 10-100 mmol / L.

8. A method for preparing the latex enhanced immunoturbidimetric detection reagent according to any one of claims 1 to 7, characterized in that: The method comprises the following steps: uniformly mixing the components of a single-reagent latex-enhanced immunoturbidimetric detection reagent according to weight percentage to obtain the latex-enhanced immunoturbidimetric detection reagent.

9. Use of the latex-enhanced immunoturbidimetric detection reagent according to any one of claims 1 to 7 in a single-reagent CRP latex-enhanced immunoturbidimetric assay kit.