WO2018129885A1 - 一种全血c反应蛋白检测试剂盒 - Google Patents

一种全血c反应蛋白检测试剂盒 Download PDF

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WO2018129885A1
WO2018129885A1 PCT/CN2017/092351 CN2017092351W WO2018129885A1 WO 2018129885 A1 WO2018129885 A1 WO 2018129885A1 CN 2017092351 W CN2017092351 W CN 2017092351W WO 2018129885 A1 WO2018129885 A1 WO 2018129885A1
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reactive protein
buffer
antibody
reagent
crp
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PCT/CN2017/092351
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French (fr)
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柴凡
冯剑军
刘兵
曹佳强
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深圳开立生物医疗科技股份有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4737—C-reactive protein

Definitions

  • the invention relates to the field of medical immunity, in particular to a whole blood C-reactive protein detection kit.
  • C-reactive protein is an acute phase protein synthesized by hepatocytes with a molecular weight of approximately 115 KD, which is present in trace amounts in healthy human serum (approximately 0.58 mg/L).
  • cytokines such as interleukin and tumor necrosis factor.
  • CRP C-reactive protein
  • CRP detection methods commonly used in clinical practice include fluorescent immunochromatography, colloidal gold method, enzyme-linked immunosorbent assay and immunoturbidimetric assay. Although the former two are sensitive, they can only be used for qualitative or semi-quantitative analysis. Although the enzyme-linked immunosorbent assay can ensure sensitivity and quantitative detection, it cannot be used for batch automated analysis. An environment with a large sample volume.
  • CRP immunoturbidimetry In the prior art, the products of CRP immunoturbidimetry are divided into two categories: common CRP and hypersensitive CRP.
  • the measurement range for ordinary CRP is generally 3-200 mg/L, and the linear range is wide but the sensitivity is low.
  • the measurement range for hypersensitive CRP is 0.3-100 mg/L, but the sensitivity is high but the high concentration CRP content cannot be determined.
  • the CRP detection kit disclosed in the patent CN105158476 and the patent CN101769932 although It can ensure high sensitivity and wide detection range at the same time, but the test samples are all serum or plasma. Because of the large amount of venous blood collected when obtaining serum or plasma, it will undoubtedly increase the suffering of the subject and is not suitable for some blood collection difficulties. Special patients, such as children, especially infants and large areas of burned people. In addition, the pretreatment of the whole blood sample increases the workload of the inspection staff, and also limits the direct automated analysis of the sample to be tested, prolonging the detection time.
  • an object of the present invention is to provide a whole blood C-reactive protein detecting kit which is tested with whole blood as a test sample, and can simultaneously ensure high sensitivity and a wide linear range.
  • the present invention adopts the following technical solutions:
  • a kit for determining C-reactive protein content in whole blood comprising reagent R1 and reagent R2, the reagent R1 comprising a hemolytic agent and a first buffer; the reagent R2 comprising an antibody-latex composite and a first Two buffers;
  • the antibody-latex complex comprises a conjugate S1 and a conjugate S2;
  • the conjugates S1 and S2 are latex particles having C-reactive protein antibodies sensitized with particle diameters of 150 nm to 250 nm and 50 nm to 150 nm, respectively;
  • the mass ratio of the conjugate S1 to the conjugate S2 is from 50:1 to 500:1.
  • the C-reactive protein antibody comprises a C-reactive protein monoclonal antibody and a C-reactive protein polyclonal antibody, wherein the conjugate S1 is a C-reactive protein monoclonal antibody-sensitized latex particle; the conjugate S2 is C Reactive protein polyclonal antibody-sensitized granule latex particles; the C-reactive protein monoclonal antibody and the C-reactive protein polyclonal antibody-sensitized latex particles have different particle sizes.
  • the kit provided by the invention uses a C-reactive protein monoclonal antibody and a C-reactive protein polyclonal antibody to respectively sensitize two specific latex particles of different particle sizes, and the latex particles of the larger average particle size are coupled with the C-reactive protein monoclonal.
  • the antibody, the latex particle with a smaller average particle size is coupled with the CRP polyclonal antibody, and then the sensitized latex particles are mixed according to a specific ratio, and the finally obtained detection kit is compared with the existing biological whole blood CRP turbidity kit. The ratio further broadens the linear range.
  • the C-reactive protein polyclonal antibody is a goat anti-human CRP polyclonal antibody, Ma Kang a CRP polyclonal antibody, a rabbit anti-human CRP polyclonal antibody or a scorpion anti-human CRP polyclonal antibody;
  • the C-reactive protein monoclonal antibody is a mouse anti-human C-reactive protein monoclonal antibody or a rabbit anti-human C-reactive protein Monoclonal antibodies.
  • the C-reactive protein monoclonal antibody-sensitized latex particles have a particle diameter of 180 to 250 nm, and the CRP polyclonal antibody-sensitized latex particles have a particle diameter of 50 to 90 nm.
  • the C-reactive protein monoclonal antibody-sensitized latex particles have a particle size of 250 nm, and the CRP polyclonal antibody-sensitized latex particles have a particle size of 70 nm.
  • the mass ratio of the conjugate S1 to the conjugate S2 is 500:1.
  • the amount of the antibody-latex complex in the reagent R2 is from 0.5 to 5 mg/ml.
  • the first buffer in the reagent R1 and the second buffer in the reagent R2 may be selected from the group consisting of a phosphate buffer solution, a Tris buffer solution, a glycine buffer solution, a Hepes buffer, a borate buffer solution, and an acetic acid solution.
  • a phosphate buffer solution a Tris buffer solution
  • a glycine buffer solution a glycine buffer solution
  • a Hepes buffer a borate buffer solution
  • an acetic acid solution acetic acid solution.
  • buffer ammonium chloride buffer or MES buffer.
  • it has a concentration of 50-500 mM and a pH of 6-8.
  • the hemolytic agent is one of Triton 100, Tween 20, Tween 80, saponin, Span 40, quaternary ammonium salt, lysophospholipid, sodium dodecyl sulfate, sodium lauryl sulfate. Or a mixture of two or more.
  • the agent R1 further comprises a polymerization inhibitor, a stabilizer, a suspending agent and a preservative.
  • the polymerization inhibitor is selected from the group consisting of PEG4000, PEG6000, PEG8000, PEG20000, dextran 20000, sodium cellulose sulfate, polyvinylpyrrolidone, and the like;
  • the stabilizer includes an ionic stabilizer and a protein stabilizer, and the ionic stabilizer is one or more of sodium chloride, sodium sulfate, calcium chloride, potassium chloride, potassium sulfate, ammonium chloride or the like. a mixture; the protein stabilizer is a mixture of one or more of bovine serum albumin, gelatin, casein, chicken serum albumin, polyamino acid;
  • the suspending agent is a mixture of one or more of glycerin, glucose, sucrose, trehalose, Triton-100, Tween 20, Tween 80;
  • the preservative is sodium azide and/or Proclin 300.
  • the stabilizer may be selected from the group consisting of one or a mixture of two or more of bovine serum albumin, chicken serum albumin, casein, gelatin; the surfactant is selected from the group consisting of Tween 20, Tween 80, and Triton 100. a mixture of one or more of NP40; a preservative may be selected from sodium azide and/or Proclin 300; in some embodiments, the stabilizer is a mixture of 1% BSA and 150 mM sodium chloride; the surfactant is 0.1% Tween 20; preservative is 0.1% sodium azide.
  • reagent R1 comprises the following components: 0.2% SDS (sodium dodecyl sulfate), 4% PEG 6000 (polyethylene glycol 6000), 200 mM sodium chloride, 2% BSA (bovine serum white) Protein), 1% glucose, 0.1% Triton X-100 (Triton 100) and 0.1% sodium azide in Tris buffer. Among them, the Tris buffer concentration was 100 mM, and the pH was 7.4.
  • Reagent R2 comprises conjugate S1, conjugate S2, pH 7.4, 100 mM Tris buffer, 1% BSA and 150 mM sodium chloride, a surfactant and a preservative, wherein the stabilizer is 1% BSA + 150 mM chlorination Sodium; surfactant is 0.1% Tween 20; preservative is 0.1% sodium azide; conjugate S1 is obtained from goat anti-human CRP polyclonal antibody sensitized with small particle size average particle size of 70 nm; The conjugate S2 was obtained by sensitizing 250 nm large latex particles with a rabbit anti-human CRP monoclonal antibody.
  • the detection kit provided by the invention has a linear detection range of 0.1-350 mg/L and a sensitivity of 0.05 mg/L, which further broadens the linear range, especially the enhanced compared with the existing biological whole blood CRP turbidity kit. Sensitivity to detection of low value samples.
  • the patient's peripheral blood or venous blood whole blood can be used as a test sample, which alleviates the blood collection pain of patients, especially newborns and burned patients, and achieves true whole blood and full-range detection for clinical hypersensitivity and common CRP samples. .
  • the whole blood full-range C-reactive protein latex-enhanced immunoturbidimetric detection kit provided by the invention can be combined with an automatic biochemical analyzer or a fully automatic specific protein analyzer and other automatic analysis instruments, which can realize batch automatic analysis and greatly improve the number of samples.
  • the detection speed shortens the time the patient waits for the test result.
  • Figure 1 shows a standard curve of the content of different concentrations of C-reactive protein calibrator (mg/mL) and its astigmatism;
  • Figure 2 is a graph showing the linear relationship between the measured value of C-reactive protein concentration and the theoretical value (mg/mL).
  • the invention discloses a whole-range full-range C-reactive protein latex-enhanced immunoturbidimetric detection kit, and those skilled in the art can learn from the contents of the paper and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application described herein may be modified or appropriately modified and combined without departing from the scope of the present invention. The technique of the present invention is applied.
  • Reagent R1 component 0.2% SDS (sodium dodecyl sulfate), 4% PEG6000 (polyethylene glycol 6000), 200 mM sodium chloride, 2% BSA (bovine serum albumin), 1% glucose, 0.1 %Triton X-100 (Traton 100) and 0.1% sodium azide in Tris buffer.
  • the Tris buffer concentration was 100 mM and the pH was 7.4.
  • Reagent R2 preparation Small particle size latex having an average particle diameter of 70 nm was sensitized with a goat anti-human CRP polyclonal antibody, and a rabbit anti-human CRP monoclonal antibody sensitized large latex particles of 250 nm. The two sensitized latex particles are resuspended by centrifugation, and diluted in a second buffer, stabilizer, surfactant, and preservative at a concentration. The above two sensitizing latex suspensions are mixed in a certain ratio to obtain a reagent R2.
  • the sensitization process is as follows (for example, JSR polystyrene latex particles having an average particle diameter of 70 nm and a latex solid content of 5%): 1 ml of latex suspension (diluted to 1% concentration with activation buffer MES) Add 20mg EDC (carbodiimide) and 20mg NHS (N-hydroxysuccinimide) in order and mix immediately, mix at room temperature by inversion or stir and stir for 15-30min; 13000rpm, centrifugation at 4 °C for 25min, remove The supernatant. Repeat twice.
  • the latex microspheres were resuspended in 1 ml of coupling buffer to form a uniformly dispersed suspension having a concentration of 1% w/v; and 1 ml of the antibody solution having a concentration of 2 mg/ml was added to the activated latex particle suspension.
  • the supernatant was transferred to another clean Ep tube, and the amount of residual antibody in the supernatant was quantified by the Bradford method, and the coupling efficiency of the antibody was calculated.
  • JSR polystyrene latex particles having an average particle diameter of 250 nm were treated in the same manner as the above-mentioned polystyrene latex particles having an average particle diameter of 70 nm.
  • the large and small latex particles were sensitized in the same manner as above, and each was diluted with a pH 7.4, 100 mM Tris buffer, 1% BSA + 150 mM sodium chloride, 0.1% Tween 20, 0.1% sodium azide. Store to a concentration of 1.5 mg/ml. Taking the above two kinds of treated latex according to the ratio of the small particle size latex to the large particle size latex of 500:1), the final reagent R2 is obtained, and the R2 reagent is a milky white uniform suspension solution.
  • Analytical method rate scattering nephelometry; instrument detection wavelength is 670 nm.
  • Calibration method 10 ul of different concentrations of calibrators (6 concentration gradients: 0 mg / L, 5 mg / L, 22.5 mg / L, 44 mg / L, 175 mg / L, 350 mg / L) mixed with 400ul R1, placed in the machine In the detection channel, 40ul R2 is added, the lowest scattering value A1 of the time period is read in the 10th second, and the scattering value A2 is read after the 60th second, and the scattering difference (ie, the final astigmatism value) of A2 minus A1 can be calculated. Subsequently, the calibration curve is calculated and plotted using the spline function (Spline). The standard curve is shown in Figure 1.
  • Whole blood sample test method Take 10ul of whole blood sample, mix it with 400ul R1, place it in the detection channel, add 40ul R2, then read the scattering values A1 and A2 after 10 seconds and 60 seconds, and calculate the difference between the two. The value is the astigmatism value that is finally read.
  • concentration of C-reactive protein in the sample can be calculated from the measured sample astigmatism value.
  • the present invention can obtain the CRP concentration in the sample to be tested within 60 seconds, and the detection linear range is as wide as 0.1-350 mg/L, which is superior to the commercially available whole blood CRP detection kit.
  • the average value of the C-reactive protein measured by the kit provided by the present invention is very close to the target value, the accuracy of the low-value sample detection is 98.36%, and the accuracy of the high-value sample detection is 99.21%, indicating that the accuracy is 99.21%.
  • the kit of invention has high accuracy.
  • the kit of the present invention has high precision.
  • the sensitivity of the kit of the present invention is extremely high, up to 0.05 mg/L.
  • High-value calibrators traceable to international standards are diluted with healthy human whole blood (without CRP) into multiple samples of different concentrations. Each sample was tested using the present invention and averaged (tested 3 times). The correlation between the test values and the theoretical dilution concentration was compared and plotted. The results are shown in Figures 2 and 4.
  • the CRP detection kit prepared according to the preparation method of the first embodiment of the present invention is different from the first embodiment in that, in the preparation of the reagent R2, a large particle having an average particle diameter of 250 nm is sensitized with a goat anti-human CRP polyclonal antibody.
  • the latex particles of the diameter, the rabbit anti-human CRP monoclonal antibody sensitize the large particle size latex particles of 250 nm, and the other steps are the same, and the detection kit of Comparative Example 1 is prepared, and then the method according to Embodiment 2 of the present invention is applied to C.
  • the reactive protein was assayed.
  • the CRP detection kit prepared according to the preparation method of the first embodiment of the present invention is different from the first embodiment in that, in the preparation of the reagent R2, a small particle having an average particle diameter of 70 nm is sensitized with a rabbit anti-human CRP monoclonal antibody.
  • the latex particles of the large diameter of the latex particles were the same in all other steps, and the test kit of Comparative Example 1 was prepared, and then the C-reactive protein was measured according to the method of Example 2 of the present invention.
  • the CRP detection kit prepared according to the preparation method of the first embodiment of the present invention is different from the first embodiment in that, in the preparation of the reagent R2, the goat anti-human CRP polyclonal antibody is used to respectively sensitize the small average particle diameter to 70 nm.
  • the latex particles of the particle size and the latex particles of the large particle size of 250 nm were the same in other steps, and the test kit of Comparative Example 1 was prepared, and then the C-reactive protein was measured according to the method of Example 2 of the present invention.
  • Example 5 of the present invention the sensitivity of the kits of Comparative Examples 1 to 3 was determined.
  • the results are shown in Tables 5-6.
  • Table 6 shows the linear detection results of Comparative Examples 1 to 3.
  • kits of Comparative Examples 1 and 3 were not as sensitive as the kit of the present invention, and the linear detection range was not as wide as the kit of the present invention.
  • the sensitivity of the kit of Comparative Example 2 was comparable to that of the kit of the present invention, the linear detection range was significantly inferior to the present invention.

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Abstract

一种全血C反应蛋白检测试剂盒。该试剂盒包括试剂R1和试剂R2，试剂R2采用C反应蛋白抗体分别致敏不同粒径的乳胶颗粒。结果表明，该试剂盒的线性检测范围宽、灵敏度高、准确性和精密度好。与现有的人全血CRP比浊试剂盒相比，进一步拓宽了线性范围，尤其是增强了对低值样本的检测灵敏度。而且，可以使用患者的末梢血或静脉血全血作为测试样本，减轻了患者尤其是新生儿及烧烫伤患者的采血痛苦，对临床超敏、普通CRP样本做到了真正的全血、全量程检测。

Description

一种全血C反应蛋白检测试剂盒
本申请要求于2017年1月13日提交中国专利局、申请号为201710026312.3、发明名称为“一种全血C反应蛋白检测试剂盒”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及医学免疫领域，特别涉及一种全血C反应蛋白检测试剂盒。
背景技术
C反应蛋白(CRP)是一种由肝细胞合成的急性时相蛋白，分子量约为115KD，在健康人体的血清中其以痕量的形式存在(约0.58mg/L)。当机体受到微生物入侵或组织损伤等炎症性刺激时，在白细胞介素、肿瘤坏死因子等细胞因子的诱导下，CRP表达量会随之急剧升高至最高达1000倍以上。而随着机体的复原，其含量则会迅速的恢复正常。因此，临床上对常规浓度CRP的检测对于鉴别细菌或病毒感染、癌症等重大疾病的进程、预后及治疗效果的评估具有广泛意义。相对于常规CRP而言，超敏C反应蛋白(浓度小于10mg/L)的临床意义除了可用于诊断低水平炎症状态之外，则主要在于对心血管疾病的风险的评判。
目前，临床常用的CRP检测方法包括荧光免疫层析、胶体金法、酶联免疫吸附法以及免疫比浊法。前两者虽然灵敏度高，但是只能做定性或半定量分析；酶联免疫吸附法检测虽然既能确保灵敏度，同时能做到定量检测，但由于方法本身无法进行批量自动化分析，因此不适用于样品量多的环境。
现有技术中，CRP免疫比浊法的产品分为对普通CRP及超敏CRP检测两大类。针对普通CRP的测定范围一般为3-200mg/L，线性范围宽但灵敏度低；而对于超敏CRP的测定范围为0.3-100mg/L，灵敏度虽高但无法测定高浓度CRP含量。
专利CN105158476及专利CN101769932公开的CRP检测试剂盒，虽然 能够同时保证高灵敏度和较宽的检测范围，但其检测样本均为血清或血浆，由于获得血清或血浆时需要大量的采集静脉血，无疑增加了受试者的痛苦且不适合某些采血困难的特殊患者，如儿童患者尤其是婴幼儿和大面积烧烫伤人群。此外对全血样本的预处理一方面增加了检验工作人员的工作量，还限制了对待测样本的直接自动化分析，延长了检测时间。
发明内容
有鉴于此，本发明目的在于提供一种全血C反应蛋白检测试剂盒，该试剂盒以全血作为测试样本进行检测，能够同时保证较高的灵敏度和较宽的线性范围。
为实现本发明的目的，本发明采用如下技术方案：
一种全血中C反应蛋白含量测定的试剂盒，其特征在于，包括试剂R1和试剂R2，所述试剂R1包括溶血剂和第一缓冲液；所述试剂R2包括抗体-乳胶复合物和第二缓冲液；
所述抗体-乳胶复合物包括偶联物S1和偶联物S2；
所述偶联物S1、S2为C反应蛋白抗体分别致敏粒径为150nm-250nm和50nm-150nm的乳胶颗粒；
所述偶联物S1和所述偶联物S2的质量比为50:1-500:1。
所述C反应蛋白抗体包括C反应蛋白单克隆抗体和C反应蛋白多克隆抗体，其中，所述偶联物S1为C反应蛋白单克隆抗体致敏的乳胶颗粒；所述偶联物S2为C反应蛋白多克隆抗体致敏的粒乳胶颗粒；所述C反应蛋白单克隆抗体和C反应蛋白多克隆抗体致敏的乳胶颗粒的粒径不同。
本发明提供的试剂盒，采用C反应蛋白单克隆抗体和C反应蛋白多克隆抗体分别致敏两种特定的不同粒径的乳胶颗粒，平均粒径较大的乳胶颗粒偶联C反应蛋白单克隆抗体，平均粒径较小的乳胶颗粒偶联CRP多克隆抗体，然后将致敏后的乳胶颗粒按照特定比例混合，最终制得的检测试剂盒与现有的生物全血CRP比浊试剂盒相比，进一步拓宽了线性范围。
优选地，C反应蛋白多克隆抗体为羊抗人CRP多克隆抗体、马抗人 CRP多克隆抗体、兔抗人CRP多克隆抗体或驴抗人CRP多克隆抗体中的一种；所述C反应蛋白单克隆抗体为鼠抗人C反应蛋白单克隆抗体或兔抗人C反应蛋白单克隆抗体。
优选地，所述C反应蛋白单克隆抗体致敏的乳胶颗粒的粒径为180～250nm，所述CRP多克隆抗体致敏的乳胶颗粒的粒径为50～90nm。在一些具体的实施例中，所述C反应蛋白单克隆抗体致敏的乳胶颗粒的粒径为250nm，所述CRP多克隆抗体致敏的乳胶颗粒的粒径为70nm。
优选地，所述偶联物S1和所述偶联物S2的质量比为500:1。
优选地，所述试剂R2中抗体-乳胶复合物的含量为0.5-5mg/ml。
本发明试剂盒中，试剂R1中的第一缓冲液和试剂R2中的第二缓冲液均可选自磷酸液缓冲液、Tris缓冲液、甘氨酸缓冲液、Hepes缓冲、硼酸盐缓冲液、醋酸缓冲液、氯化铵缓冲液或MES缓冲液中的一种。优选地，其浓度为50-500mM，pH为6-8。
所述溶血剂为曲拉通100、吐温20、吐温80、皂素、司盘40、季铵盐、溶血磷脂、十二烷基磺酸钠、十二烷基硫酸钠中的一种或两者以上的混合物。
优选地，所述试剂R1中还包括促聚剂、稳定剂、助悬剂和防腐剂。其中，促聚剂选自PEG4000、PEG6000、PEG8000、PEG20000、葡聚糖20000、硫酸纤维素钠、聚乙烯吡咯烷酮等中的一种或两者以上的混合物；
稳定剂包括离子型稳定剂和蛋白类稳定剂，所述离子型稳定剂为氯化钠、硫酸钠、氯化钙、氯化钾、硫酸钾、氯化铵中的一种或两者以上的混合物；所述蛋白类稳定剂为牛血清白蛋白、明胶、酪蛋白、鸡血清白蛋白、多聚氨基酸中的一种或两者以上的混合物；
助悬剂为甘油、葡萄糖、蔗糖、海藻糖、曲拉通-100、吐温20、吐温80等一种或两者以上的混合物；
防腐剂为叠氮化钠和/或Proclin300。
优选地，R2中还包括稳定剂、表面活性剂和防腐剂。其中，稳定剂可以选自牛血清白蛋白、鸡血清白蛋白、酪蛋白、明胶中的一种或两者以上的混合物；表面活性剂选自吐温20、吐温80，曲拉通100，NP40中的一种或两者以上的混合物；防腐剂可选叠氮化钠和/或Proclin300；在一些具体实施例中，稳定剂为1％BSA和150mM氯化钠的混合物；表面活性剂为0.1％吐温20；防腐剂为0.1％的叠氮化钠。
在一些具体实施方案中，试剂R1包含如下组分：0.2％SDS(十二烷基磺酸钠)、4％PEG6000(聚乙二醇6000)、200mM氯化钠、2％BSA(牛血清白蛋白)、1％葡萄糖、0.1％Triton X-100(曲拉通100)以及0.1％叠氮化钠的Tris缓冲液。其中，Tris缓冲液浓度为100mM，pH为7.4。
试剂R2包含偶联物S1、偶联物S2、pH 7.4,100mM的Tris缓冲液、1％BSA和150mM氯化钠、表面活性剂和防腐剂，其中，稳定剂为1％BSA+150mM氯化钠；表面活性剂为0.1％吐温20；防腐剂为0.1％的叠氮化钠；偶联物S1由羊抗人CRP多克隆抗体致敏平均粒径为70nm的小粒径乳胶获得；偶联物S2由兔抗人CRP单克隆抗体致敏250nm的大乳胶颗粒获得。
本发明提供的检测试剂盒的线性检测范围为0.1-350mg/L，灵敏度为0.05mg/L，与现有的生物全血CRP比浊试剂盒相比，进一步拓宽了线性范围，尤其是增强了对低值样本的检测灵敏度。同时，可以使用患者的末梢血或静脉血全血作为测试样本，减轻了患者尤其是新生儿及烧烫伤患者的采血痛苦，对临床超敏、普通CRP样本做到了真正的全血、全量程检测。而且，本发明提供的全血中全量程C反应蛋白乳胶增强免疫比浊检测试剂盒能够搭配全自动生化仪或全自动特定蛋白仪及其他自动分析仪器，能够实现批量自动化分析，极大地提高了检测速度，缩短了患者等待检测结果的时间。
附图说明
图1示不同浓度C反应蛋白校准品的含量(mg/mL)及其散光度建立的标准曲线图；
图2示C反应蛋白浓度检测值与理论值(mg/mL)的线性关系图。
具体实施方式
本发明公开了一种全血中全量程C反应蛋白乳胶增强免疫比浊检测试剂盒，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
下面结合实施例，进一步阐述本发明：
实施例1全血全量程CRP检测试剂盒的制备
试剂R1组分:含0.2％SDS(十二烷基磺酸钠)、4％PEG6000(聚乙二醇6000)、200mM氯化钠、2％BSA(牛血清白蛋白)、1％葡萄糖、0.1％Triton X-100(曲拉通100)以及0.1％叠氮化钠的Tris缓冲液。Tris缓冲液浓度为100mM，pH为7.4。
试剂R2制备：用羊抗人CRP多克隆抗体致敏平均粒径为70nm的小粒径乳胶，兔抗人CRP单克隆抗体致敏250nm的大乳胶颗粒。两种致敏的乳胶颗粒经离心漂洗后超声重悬，并按照一定的浓度稀释于第二缓冲液、稳定剂、表面活性剂和防腐剂中备用。上述两种致敏胶乳悬浮液按照一定的比例进行混合，即得到试剂R2。
所述的致敏过程如下(以平均粒径为70nm，胶乳固含量为5％的JSR聚苯乙烯乳胶颗粒为例叙述)：向1ml胶乳悬浮液(已用活化缓冲液MES稀释至1％浓度)中按顺序加入20mgEDC(碳二亚胺)及20mg NHS(N-羟基琥珀酰亚胺)并立即混匀，室温颠倒混匀或搅拌混匀活化15-30min；13000rpm,4℃离心25min，去除上清。重复两次。随后以1ml偶联缓冲液重悬乳胶微球，使其成为均匀分散，浓度为1％w/v的悬液；向上述已活化的乳胶颗粒悬液中加入1ml浓度为2mg/ml的抗体溶液并迅速搅拌均匀，室温下搅拌孵育2h；加入终浓度为10mM的盐酸羟胺终止偶联反应； 13000rpm，4℃离心20min。将上清转移至另一干净的Ep管中，用Bradford法定量上清中残余抗体的数量，并计算抗体的偶联效率。再加入1ml偶联缓冲液重悬洗涤并按上述条件再次离心；以生理盐水(含1％BSA)或PBS缓冲液(pH 7.2；另含1％BSA)重悬抗体-乳胶颗粒，使其均匀分散在溶液中备用，如需要可进行超声分散。
平均粒径为250nm的JSR聚苯乙烯乳胶颗粒和上述平均粒径为70nm的聚苯乙烯乳胶颗粒的处理方法相同。
大、小乳胶颗粒按照上述同样的方法进行致敏后，各自以pH7.4,100mM浓度的Tris缓冲液、1％BSA+150mM氯化钠、0.1％吐温20、0.1％的叠氮化钠稀释至浓度为1.5mg/ml储存。取上述两种处理后的胶乳按照小粒径胶乳与大粒径胶乳比例为500:1)，即可得到最终的试剂R2，R2试剂为乳白色均匀的悬浮溶液。
实施例2 C反应蛋白的测定
检测工具：开立半自动特定蛋白仪
分析方法：速率散射比浊法；仪器检测波长为670nm。
校准方法：将10ul不同浓度的校准品(6个浓度梯度：0mg/L、5mg/L、22.5mg/L、44mg/L、175mg/L、350mg/L)与400ul R1混合，放置在机器的检测通道内，再加入40ul R2，第10秒读取该时间段最低散射值A1，第60秒后读取散射值A2，即可计算出A2减A1的散射差值(即最终散光值)。随后，利用样条函数(Spline)对校准品浓度和散射值进行校准曲线的计算和绘制，标准曲线如图1所示。
全血样本测试方法：取全血样本10ul，与400ul R1混合后放置于检测通道中，再加入40ul R2，随后依次在10秒和60秒后读取散射值A1和A2，计算二者的差值即为最终读取的散光值。利用上述定标的校准曲线和标准曲线方程，即可依据测得的样本散光值计算得到样本中C反应蛋白的浓度。
不考虑加样时间，本发明在60秒内即可获得待测样本中的CRP浓度，且检测线性范围宽达0.1-350mg/L,均优于市面上的全血CRP检测试剂盒。
实施例3本发明试剂盒准确度分析
取可溯源至国际标准的血清校准品两支，包含高值和低值各一支。利用本发明的试剂及本公司特定蛋白仪对相关样品进行测试，每支样品重复检测10次，并与校准品靶值进行比较。结果见表1：
表1准确度检测结果
Figure PCTCN2017092351-appb-000001
由表1数据可知，本发明提供的试剂盒测定的C反应蛋白的平均值与靶值很接近，低值样品检测的准确度为98.36％，高值样品检测的准确度为99.21％，表明本发明试剂盒准确度高。
实施例4本发明试剂盒精密度分析
取全血样品两支，包含CRP含量高值和低值各一支。利用本发明的试剂及本公司特定蛋白仪对相关样品进行测试，每支样品重复检测10次。计算检测结果的平均值、标准差和变异系数(CV)，结果见表2。
表2精密度检测结果
测试次数 样品1(低值，单位mg/L) 样品2(高值，单位mg/L)
1 20.62 144.5
2 20.47 145
3 19.4 143.75
4 20.24 144.06
5 20.73 144.27
6 20.48 147.12
7 20.4 143.84
8 21.7 145.39
9 19.84 144.11
10 20.24 143.12
平均值 20.412 144.47
标准差 0.59 1.06
变异系数 2.89％ 0.73％
结果显示，低值样品标准差率为0.59％，高值样品标准差率为1.06，均小于标准的3％；低值样品CV＝2.89％，高值样品CV＝0.73％，均小于5％，符合《体外诊断试剂通用要求》表明本发明所述试剂盒具有高精密度。
实施例5本发明试剂盒灵敏度分析
取不含CRP的健康人全血样本一只，利用本发明试剂对其进行测试，重复检测10次并依据平均值和标准差计算灵敏度，结果见表3。
表3灵敏度检测结果
Figure PCTCN2017092351-appb-000002
Figure PCTCN2017092351-appb-000003
由表3数据可知，本发明试剂盒的灵敏度极高，可达0.05mg/L。
实施例6本发明试剂盒线性的检测
取可溯源至国际标准的高值校准品，用健康人全血(不含CRP)稀释成多个不同浓度的样本。对每个样本利用本发明进行测试并取平均值(测试3次)。将测试数值与理论稀释浓度进行相关性比较并作图，结果见图2和表4。
表4线性分析结果
Figure PCTCN2017092351-appb-000004
Figure PCTCN2017092351-appb-000005
由图2和表4的结果可知，在0.1-350mg/L范围内，R2＝0.9944，表明本发明试剂盒检测C反应蛋白的线性好，检测范围宽。
实施例7
对比例1
按照本发明实施例1制备方法制备所述的CRP检测试剂盒，与实施例1的区别在于，在试剂R2的制备中，用羊抗人CRP多克隆抗体致敏平均粒径为250nm的大粒径的乳胶颗粒，兔抗人CRP单克隆抗体致敏250nm的大粒径的乳胶颗粒，其他步骤条件均相同，制备得到对比例1的检测试剂盒，然后按照本发明实施例2的方法对C反应蛋白进行测定。
对比例2
按照本发明实施例1制备方法制备所述的CRP检测试剂盒，与实施例1的区别在于，在试剂R2的制备中，用兔抗人CRP单克隆抗体致敏平均粒径为70nm的小粒径的乳胶颗粒大粒径的乳胶颗粒，其他步骤条件均相同，制备得到对比例1的检测试剂盒，然后按照本发明实施例2的方法对C反应蛋白进行测定。
对比例3
按照本发明实施例1制备方法制备所述的CRP检测试剂盒，与实施例1的区别在于，在试剂R2的制备中，用羊抗人CRP多克隆抗体分别致敏平均粒径为70nm的小粒径的乳胶颗粒和250nm的大粒径的乳胶颗粒，其他步骤条件均相同，制备得到对比例1的检测试剂盒，然后按照本发明实施例2的方法对C反应蛋白进行测定。
按照本发明实施例5的方法，对对比例1～3的试剂盒进行灵敏度分 析和线性范围的检测，结果见表5～6。
表5对比例1～3的灵敏度检测结果
  平均值(M) 标准差(SD) 灵敏度(M+2.6SD)
对比例1 0.08 0.002 0.0852
对比例2 0.0498 0.002 0.055
对比例3 0.1001 0.0032 0.1084
表6对比例1～3的线性检测结果
  线性范围 R2
对比例1 0.3-280mg/ml 0.992
对比例2 0.12-180mg/ml 0.994
对比例3 0.4-350mg/ml 0.985
结果显示，对比例1、3的试剂盒灵敏度均不如本发明试剂盒高，线性检测范围也不如本发明试剂盒宽。虽然对比例2的试剂盒的灵敏度与本发明试剂盒相当，但其线性检测范围明显不如本发明宽。
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种全血中C反应蛋白含量测定的试剂盒，其特征在于，包括试剂R1和试剂R2，所述试剂R1包括溶血剂和第一缓冲液；所述试剂R2包括抗体-乳胶复合物和第二缓冲液；
    所述抗体-乳胶复合物包括偶联物S1和偶联物S2；
    所述偶联物S1、S2为C反应蛋白抗体分别致敏的粒径为150nm-250nm和50nm-150nm的乳胶颗粒；
    所述偶联物S1和所述偶联物S2的质量比为50:1-500:1。
  2. 根据权利要求1所述的试剂盒，其特征在于，所述C反应蛋白抗体包括C反应蛋白单克隆抗体和C反应蛋白多克隆抗体，其中，所述偶联物S1为C反应蛋白单克隆抗体致敏的乳胶颗粒；所述偶联物S2为C反应蛋白多克隆抗体致敏的粒乳胶颗粒；所述C反应蛋白单克隆抗体和C反应蛋白多克隆抗体致敏的乳胶颗粒的粒径不同。
  3. 根据权利要求1所述的试剂盒，其特征在于，所述C反应蛋白多克隆抗体为羊抗人CRP多克隆抗体、马抗人CRP多克隆抗体、兔抗人CRP多克隆抗体或驴抗人CRP多克隆抗体中的一种；所述C反应蛋白单克隆抗体为鼠抗人C反应蛋白单克隆抗体或兔抗人C反应蛋白单克隆抗体。
  4. 根据权利要求1所述的试剂盒，其特征在于，所述C反应蛋白单克隆抗体致敏的乳胶颗粒的粒径为180～250nm，所述CRP多克隆抗体致敏的乳胶颗粒的粒径为50～90nm。
  5. 根据权利要求1所述的试剂盒，其特征在于，所述试剂R2中抗体-乳胶复合物的含量为0.5-5mg/ml。
  6. 根据权利要求1所述的试剂盒，其特征在于，所述第一缓冲液、第二缓冲液选自磷酸液缓冲液、Tris缓冲液、甘氨酸缓冲液、Hepes缓冲、硼酸盐缓冲液、醋酸缓冲液、氯化铵缓冲液或MES缓冲液中的一种。
  7. 根据权利要求6所述的试剂盒，其特征在于，所述第一缓冲液和第二缓冲液的浓度为50-500mM，pH为6-8。
  8. 根据权利要求1所述的试剂盒，其特征在于，所述溶血剂为曲拉通100、吐温20、吐温80、皂素、司盘40、季铵盐、溶血磷脂、十二烷基磺酸钠、十二烷基硫酸钠中的一种或两者以上的混合物。
  9. 根据权利要求1所述的试剂盒，其特征在于，所述试剂R1中还包括促聚剂、稳定剂、助悬剂和防腐剂。
  10. 根据权利要求1所述的试剂盒，其特征在于，所述试剂R2中还包括稳定剂、表面活性剂和防腐剂。
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