阅读说明：本技术 一种全量程c反应蛋白检测试剂盒 (Full-range C reactive protein detection kit ) 是由 戴瞻 于 2019-10-10 设计创作，主要内容包括：一种全量程c反应蛋白检测试剂盒,通过免疫透射比浊法,检测样本中c反应蛋白的含量,达到定量检测的目的。该试剂盒包含试剂R1、试剂R2,所述的试剂R1为磷酸盐缓冲溶液,试剂R2为两种大小粒径的胶乳分别与两种抗人c反应蛋白抗体交联形成的偶联物。同时,该试剂盒与全自动生化分析仪联合使用,提高了检测的灵敏度与准确度,缩短了检测时间。本发明的全量程C反应蛋白检测试剂盒既可以测定高浓度CRP含量,又可以测定低浓度CRP含量,且灵敏度高、稳定性强、线性范围宽、重复性好,具有良好的抗干扰性、准确度和精密度,能够满足临床检验要求。(A full-range c-reactive protein detection kit detects the content of c-reactive protein in a sample by an immune transmission turbidimetry method to achieve the aim of quantitative detection. The kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 is phosphate buffer solution, and the reagent R2 is a conjugate formed by respectively crosslinking latex with two types of anti-human c-reactive protein antibodies in two sizes. Meanwhile, the kit is used together with a full-automatic biochemical analyzer, so that the sensitivity and the accuracy of detection are improved, and the detection time is shortened. The full-range C-reactive protein detection kit can be used for determining the content of high-concentration CRP and low-concentration CRP, has the advantages of high sensitivity, strong stability, wide linear range and good repeatability, has good anti-interference performance, accuracy and precision, and can meet the requirements of clinical examination.)

1. A full-scale C-reactive protein detection kit is characterized in that: the kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises 50m of phosphate buffer solution, 0.1 percent of tween-20, 0.1 percent of BSA, 2 percent of PEG-6000 and 0.1 percent of preservative; the reagent R2 is a conjugate formed by respectively crosslinking two latex microspheres with large and small particle sizes with two anti-human CRP monoclonal antibodies; the latex microspheres are carboxyl modified carboxylated polystyrene latex microspheres; the cross-linking mode of the anti-human CRP monoclonal antibody and the carboxylated polystyrene latex microspheres is the combination of covalent cross-linking and chemical cross-linking;

the preparation method of the reagent R2 is as follows: respectively sensitizing two kinds of carboxylated polystyrene latex microspheres with different large and small particle sizes by two kinds of anti-human CRP monoclonal antibodies, wherein the average particle size range of the carboxylated polystyrene latex microspheres with large particle sizes is 200-400nm, the average particle size range of the carboxylated polystyrene latex microspheres with small particle sizes is 50-200nm, respectively dissolving the carboxylated polystyrene latex microspheres with large particle sizes and the carboxylated polystyrene latex microspheres with small particle sizes in a fatty acid methyl ester sulfonate reaction solution for mixing, then adding an EDC solution, and adding 0.1mg/mLEDC solution into 10mg of latex mass for preparation, thereby obtaining a reagent R2.

2. The full-scale C-reactive protein detection kit according to claim 1, characterized in that: the average particle size range of the large-particle-size carboxylated polystyrene latex microspheres is 250-350 nm; the average particle size range of the small-particle-size carboxylated polystyrene latex microspheres is 60-100 nm.

3. The full-scale C-reactive protein detection kit according to claim 1, characterized in that: the kit also comprises a C-reactive protein standard substance, wherein the content of the C-reactive protein in the C-reactive protein standard substance is 0.1-300 mg/L.

4. The full-scale C-reactive protein detection kit according to claim 1, characterized in that: the reagent R1 and the reagent R2 are both stable for 12 months in closed storage at the temperature of 2-8 ℃.

5. The use of the full-scale C-reactive protein detection kit of claim 1 to detect the concentration of full-scale C-reactive protein.

6. The use of claim 5, wherein, in use, the full-scale C-reactive protein detection kit is used in combination with a full-automatic biochemical analyzer, and in the detection process, the sample dosage is 2.1 μ L, and the detection time is 5 min; the detection steps are as follows: uniformly mixing 2.1 mu L of sample and 210 mu L of reagent R1, incubating at 37 ℃ for 3min, reading the 1 st reading point absorbance A1 at 600nm, immediately adding 70 mu L of reagent R2, uniformly mixing, incubating at 37 ℃ for 5min, and reading the 2 nd reading point absorbance A2 at 600 nm; and (3) taking the absorbance difference delta A-A2-A1 as a y axis and the concentration value of the calibrator as an x axis, drawing a calibration curve in a multipoint nonlinear calibration mode, and calculating the obtained value according to the calibration curve to obtain the content of the C-reactive protein in the sample.

Technical Field

The invention belongs to the field of medical immunity, relates to a full-range C-reactive protein detection kit, and particularly relates to a full-range C-reactive protein latex enhanced immunoturbidimetric detection kit.

Background

C-reactive protein (CRP) is an acute phase-reactive protein, which is secreted from the liver when the body is stimulated by invasion of microorganisms or tissue damage, and rapidly increases within several hours, but its content rapidly decreases in the disease recovery period with the restoration of tissue structure and function, and thus has important clinical significance in early diagnosis, disease course, prognosis, and the like of diseases such as infection, inflammation, and the like. The generation mechanism is as follows: when the body is infected or the tissues are damaged, macrophages and other white blood cells are activated to produce cytokines such as interleukin-6 (IL-6), interleukin-l (IL-l), tumor necrosis factor (TNF-a) and other mediators, and the cytokines and the mediators reach the liver to stimulate the liver cells and the epithelial cells to synthesize CRP. Depending on the sensitivity of the detection method, C-reactive protein can be classified into common CRP, hypersensitive CRP (hs-CRP) and full-scale CRP. The common CRP is mainly used for evaluating infection, tissue injury and inflammatory diseases clinically and providing information for diagnosis, treatment and monitoring of the inflammatory diseases. Hypersensitive CRP is a sensitive index for diagnosing low-level inflammatory states and plays an important role in the assessment of cardiovascular disease risks. The full-scale CRP can be used for diagnosis and treatment effect monitoring of infectious diseases, can also be used as an independent predictor of cardiovascular diseases and can be used for evaluating the degree of disease infection.

The full-range CRP is named because its measurement method is more sensitive and the measurement range is broader. The clinical conventional CRP measuring method is an immunoturbidimetry method, the measuring range is generally 3-200 mg/L, the linear range is wide, but the sensitivity is low, and the CRP content below the level of 3mg/L cannot be accurately measured. The clinical hypersensitive CRP measuring range is 0.3-100 mg/L, although the sensitivity is high, the CRP content at a high concentration level cannot be accurately measured, and therefore, the clinical hypersensitive CRP measuring range cannot be used for diagnosis or curative effect observation of infectious diseases.

At present, there are a variety of CRP detection reagents used clinically. For example, the C-reactive protein detection kit disclosed in chinese patent with publication number CN 103941017B, the full-scale C-reactive protein detection kit disclosed in chinese patent with publication numbers CN 105158476B and CN 101769932B, and the like. Although the C-reactive protein detection kits disclosed by CN 105158476B and CN 101769932B in the detection kits can simultaneously ensure high sensitivity and a wider detection range, the preparation process of R2 in the detection kit used in the detection kits adopts a two-step method, and 1-2 days are required for completing the coating of the antigen monoclonal antibody to be detected on the latex microsphere, so that the operation steps are increased, the time and the labor are wasted, the requirement on raw materials is increased, the sensitivity is not high, and the production cost is increased.

Therefore, how to solve the above problems is an important research content for those skilled in the art.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide a full-range C-reactive protein detection kit, which is a full-range C-reactive protein latex enhanced immunoturbidimetric detection kit, can be used for measuring the content of high-concentration C-reactive protein and the content of low-concentration C-reactive protein, and has the advantages of high sensitivity, strong stability, wide linear range, good repeatability and low price.

In order to achieve the above and other related objects, the present invention provides a full-scale C-reactive protein assay kit, comprising a reagent R1 and a reagent R2, wherein the reagent R1 comprises the following components, 50m phosphate buffer solution, 0.1% tween-20, 0.1% BSA, 2% PEG-6000, and 0.1% preservative; the reagent R2 is a conjugate formed by respectively crosslinking two latex microspheres with large and small particle sizes with two anti-human CRP monoclonal antibodies; the latex microspheres are carboxyl modified carboxylated polystyrene latex microspheres; the cross-linking mode of the anti-human CRP monoclonal antibody and the carboxylated polystyrene latex microspheres is the combination of covalent cross-linking and chemical cross-linking;

the preparation method of the reagent R2 is as follows: respectively sensitizing two kinds of carboxylated polystyrene latex microspheres with different large and small particle sizes by two kinds of anti-human CRP monoclonal antibodies, wherein the average particle size range of the carboxylated polystyrene latex microspheres with large particle sizes is 200-400nm, the average particle size range of the carboxylated polystyrene latex microspheres with small particle sizes is 50-200nm, respectively dissolving the carboxylated polystyrene latex microspheres with large particle sizes and the carboxylated polystyrene latex microspheres with small particle sizes in fatty acid methyl ester sulfonate reaction liquid, mixing, adding EDC solution, and adding 0.1mg/mLEDC solution into 10mg of latex mass to prepare the reagent R2.

Further, the reaction solution of the anti-human CRP monoclonal antibody and MES was mixed in a ratio of 1 mg: mixing 0.5mL of the mixture, and then placing the mixture on a vortex mixer to mix for more than 10S; mixing the carboxylated polystyrene latex microspheres and the MES reaction solution, and then putting the mixture on a vortex mixer to mix for more than 10 seconds; mixing the EDC reaction liquid and the MES reaction liquid, and then placing the mixture on a vortex mixer to mix for more than 10S; adding the carboxylated polystyrene latex microspheres into an anti-human CRP monoclonal antibody, uniformly mixing for 10S on a vortex mixer, adding the prepared EDC solution into the mixed solution, and uniformly mixing for 10S on the vortex mixer; mix well at room temperature for 3 h.

Furthermore, 10% BSA was taken from each tube and dissolved in purified water, and then added to the microspheres for blocking to prevent non-specific aggregation of the microspheres.

Further, the average particle size range of the large-particle-size carboxylated polystyrene latex microspheres is 250-350 nm; more preferably, the particle size is 278 nm. The average particle size range of the small-particle-size carboxylated polystyrene latex microspheres is 60-100 nm; more preferably, the particle size is 60nm, so that the prepared detection kit can ensure that the antigen-antibody reaction test can be performed in a wider range and the stable time can be prolonged. The average particle size of the carboxylated polystyrene latex microspheres with different particle sizes is 50-400nm, when the particle size is less than 50nm, the absorbance change caused by the aggregation of latex particles is very small, and the requirement on test sensitivity is difficult to realize as a result, but when the latex particles with the particle size of more than 400nm are used for measuring a high-concentration detection object, the absorbance change caused by the aggregation exceeds the detection limit, and the self-aggregation is accelerated and the dispersity is reduced due to larger particles.

Furthermore, the large and small particle size carboxylated polystyrene microspheres are activated and coupled to the surfaces of the microspheres by the anti-human CRP monoclonal antibody through EDC solution to form stable amido bonds. And then sealing and cleaning, and storing the prepared microspheres in a specific buffer solution at 4 ℃. The reagent performance was then tested using a fully automated biochemical analyzer.

Further, the full-scale C-reactive protein detection kit also comprises a C-reactive protein standard substance, wherein the content of C-reactive protein in the C-reactive protein standard substance is 0.1-300 mg/L.

Further, the reagent R1 and the reagent R2 are both stable for 12 months in closed storage at the temperature of 2-8 ℃.

The invention also provides application of the full-range C-reactive protein detection kit in detection of the concentration of the full-range C-reactive protein.

Further, when in use, the full-range C-reactive protein detection kit is used in combination with a full-automatic biochemical analyzer, in the detection process, the sample dosage is 2.1 muL, and the detection time is 5 min; the detection steps are as follows: uniformly mixing 2.1 mu L of sample and 210 mu L of reagent R1, incubating at 37 ℃ for 3min, reading the 1 st reading point absorbance A1 at 600nm, immediately adding 70 mu L of reagent R2, uniformly mixing, incubating at 37 ℃ for 5min, and reading the 2 nd reading point absorbance A2 at 600 nm; and (3) taking the absorbance difference delta A-A2-A1 as a y axis and the concentration value of the calibrator as an x axis, drawing a calibration curve in a multipoint nonlinear calibration mode, and calculating the obtained value according to the calibration curve to obtain the content of the C-reactive protein in the sample.

The detection principle of the invention is as follows: the latex enhanced immunoturbidimetry method adopted by the invention uses a mode of combining chemical crosslinking and covalent crosslinking, so that the reaction is faster, the reagent is more stable, and the sensitivity is higher. The carboxylated polystyrene latex microspheres with small particle size can ensure the linearity of the reagent, and the carboxylated polystyrene latex microspheres with large particle size can ensure that the reagent has higher sensitivity and can detect the content of the detected object in a low concentration range. The invention adopts conjugates formed by respectively crosslinking two carboxylated polystyrene latex microspheres with different particle sizes and two different anti-human CRP monoclonal antibodies as a reagent R2, and jointly uses latex with large and small particle sizes, so that the sensitivity and linearity of the reagent are improved.

Due to the application of the technical scheme, compared with the prior art, the invention has the following beneficial effects:

(1) the invention respectively uses the conjugate formed by crosslinking two carboxylated polystyrene latex microspheres with different particle sizes and two anti-human CRP monoclonal antibodies as the reagent R2, so that the reagent ensures high sensitivity and simultaneously ensures the linear range, and simultaneously widens the detection range of the reagent. The common C reactive protein detection kit is limited to detect the content of C reactive protein of more than 3mg/L, and cannot be used for predicting the risk of cardiovascular diseases and the detection of local infection or small focus in the future. However, the kit of the invention not only can accurately measure the content of C-reactive protein with the level of less than 3mg/L, the sensitivity can reach 0.1mg/L, and the kit can be used for risk prediction of cardiovascular diseases and detection of local infection, but also can measure the content of C-reactive protein with high concentration level (the content is as high as 300mg/L), so that the kit can be used for diagnosing various infectious and inflammatory diseases and the like.

(2) The detection kit has high detection sensitivity. The reaction sensitivity is improved by the mixture ratio of the reagent buffer solution, the surfactant and the like.

(3) The detection kit has high accuracy. The correlation with the control reagent serum full-scale C-reactive protein detection reagent reaches 0.990.

(4) The reagent of the detection kit is stable. The mixture ratio of the components of the reagent is optimized, and the reagents R1 and R2 can be stably stored under the condition of 2-8 ℃ in a sealed way for 12 months.

(5) The detection kit can be used for batch detection. The device is suitable for a full-automatic biochemical analyzer and can realize rapid and batch detection.

(6) The method for covalently bonding the microspheres with the antibody adopts a one-step method, and saves time and reduces cost compared with a two-step method.

(7) The invention is suitable for a wide range of full-automatic biochemical analysis analyzers. The types are as follows: hitachi 7080/7180/7600 olympus AU480/640/680/5400/5800 and the like.

Drawings

FIG. 1 is a schematic diagram showing the measurement results of the sensitivity of the full-scale CRP detection kit (60nm and 278nm microsphere 3:1 coated kit) of the present invention;

FIG. 2 is a graph showing the results of the precision of the CRP assay reagent of the present invention and a CRP assay reagent of a foreign known brand.

Detailed Description

Other advantages and capabilities of the present invention will be readily apparent to those skilled in the art from the disclosure of the present specification by describing the embodiments of the present invention with reference to the specific embodiments thereof.