WO2019011125A1 - 一种用于酶联免疫试剂盒的组合物以及幽门螺旋杆菌抗体谱检测试剂盒及其制备方法 - Google Patents

一种用于酶联免疫试剂盒的组合物以及幽门螺旋杆菌抗体谱检测试剂盒及其制备方法 Download PDF

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WO2019011125A1
WO2019011125A1 PCT/CN2018/093060 CN2018093060W WO2019011125A1 WO 2019011125 A1 WO2019011125 A1 WO 2019011125A1 CN 2018093060 W CN2018093060 W CN 2018093060W WO 2019011125 A1 WO2019011125 A1 WO 2019011125A1
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enzyme
kit
helicobacter pylori
bsa
citric acid
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PCT/CN2018/093060
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English (en)
French (fr)
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王洪涛
张永顶
马伟民
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深圳市伯劳特生物制品有限公司
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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates

Definitions

  • the invention relates to the field of enzyme-linked immunoassay, in particular to a composition for an enzyme-linked immunoassay kit and a Helicobacter pylori antibody spectrum detecting kit and a preparation method thereof.
  • H. pylori is a pathogen that colonizes the gastric mucosa and has an infection rate of up to 50-70% worldwide.
  • the bacteria can cause a variety of digestive diseases, and may have some connection with certain diseases other than the digestive tract, such as anemia, indigestion and some autoimmune diseases.
  • H.pylori The pathogenicity and carcinogenicity of H.pylori depend on its virulence factors.
  • virulence factors are Cytotoxin associated protein A (CagA), Vacaolating toxin A (VacA), Urease A (UreA), Urease B (UreB), Heat Shock Protein 60 (HSP60), ⁇ -glutamyl transpeptidase (gGT), flagella hook Related proteins (Flagellar hook-associated protein 2, FliD), high temperature requirement A (HtrA), Helicobacter pylori adhesin (HpaA), Cell translocating kinase (Cell translocating kinase, CtkA), Neutrophil-activating protein (NapA), hypothetical protein (HP231), gastric cancer related factor (JHP940), outer membrane protein (Omp), cysteine-rich protein C (HcpC), Catalase, an iron-dependent carrier transporter (TonB Protein).
  • CagA Cy
  • H. pylori have the above virulence factors, and the different virulence factors and quantities of different H. pylori strains also determine the virulence, pathogenicity and carcinogenic risk of the strain itself. It indicated that CagA, VacA and GroEL (HSP60) are potential markers for identifying high-risk H. pylori strains in patients, which are related to the occurrence of gastric cancer and can be used as a risk stratification index for H. pylori gastric cancer. Infection with H. pylori strains carrying different virulence factors results in the body producing antibodies against different virulence factors.
  • analysis of antibody composition and titer in the serum of infected persons can help reflect the infection of H. pylori in infected persons.
  • the type and quantity of virulence factors possessed by the strain thereby predicting the virulence and pathogenicity and clinical consequences of H. pylori, selecting an appropriate treatment plan, reflecting the spirit of precision medicine, and reducing the risk and cost of treatment.
  • ELISA enzyme-linked immunosorbent assay
  • a solid phase carrier such as polystyrene
  • ELISA enzyme-linked immunosorbent assay
  • Chinese patent CN102721815A provides a kit for detecting Helicobacter pylori virulence protein antibody, which is based on the principle of ELISA, and uses six Helicobacter pylori virulence proteins as antigens to detect the serum of patients, thereby judging the virulence of the strain and realizing it.
  • Semi-quantitative testing However, the stability of the kit is not good, which is a common problem faced by all types of enzyme-linked immunoassay kits. In addition, the kit still cannot distinguish between different subtypes of H. pylori, and the detection specificity and sensitivity are also Not quite satisfactory.
  • an object of the present invention is to provide a composition for an enzyme-linked immunoassay kit, which enables the composition to significantly improve the stability of the kit at low temperature and room temperature when used in the preparation of an enzyme-linked immunoassay kit. , the storage time is extended;
  • Another object of the present invention is to provide an application of the above composition in the preparation of an enzyme-linked immunoassay kit, particularly a kit for detecting H. pylori;
  • Another object of the present invention is to provide a Helicobacter pylori antibody spectrum detecting kit comprising the above composition and a preparation method thereof, which have the stability of the kit at a low temperature and a room temperature for a long period of time, and the detection result has Higher specificity and sensitivity.
  • the present invention provides the following technical solutions:
  • a composition for an enzyme-linked immunoassay kit comprising a blocking solution and an enzyme standard dilution; the blocking solution containing BSA, betaine, mannitol, sodium azide, disodium hydrogen phosphate and citric acid, the enzyme
  • the standard dilutions contain Tris, citric acid, BSA, gum arabic, betaine and Proclin300.
  • the present invention unexpectedly finds that the blocking solution and the enzyme standard dilution solution (for diluting the enzyme-labeled antigen or antibody) of the preparation kit are used. Selecting the appropriate components to improve the composition of both can significantly improve the stability and storage time of the enzyme-linked immunoassay kit.
  • the blocking solution contains 0.6% to 1% BSA, 0.8% to 1.5% betaine, 0.8% to 1.5% mannitol, 0.02% sodium azide, 0.01 M disodium hydrogen phosphate, and 1.5% to 2.5%.
  • the enzyme standard dilution contains 0.1 M Tris, 0.05 M citric acid, 2%-3% BSA, 1.8%-2.4% gum arabic, 0.8%-1.5% betaine, and 0.05% Proclin300, the balance is water, except for Proclin300, which is a percentage by volume, the rest is mass percentage (w/v); in a specific embodiment of the present invention, the enzyme standard dilution contains 0.1M Tris, 0.05 M citric acid, 2.5% BSA, 2% gum arabic, 1% betaine, and 0.05% Proclin 300.
  • the invention utilizes the above composition to prepare the Helicobacter pylori enzyme-linked immunoassay kit, and compares with the conventional blocking solution and the enzyme standard dilution Helicobacter pylori enzyme immunoassay kit, at a low temperature (2-8 ° C),
  • the kit of the present invention was able to maintain stability upon completion of preparation after 24 months of storage, while the control kit had a large degree of instability after 18 months of storage; at room temperature (18-28 ° C)
  • the kit of the present invention was able to maintain stability upon completion of preparation after 12 months of standing, while the control kit showed a large degree of instability after 6 months of standing.
  • the present invention proposes the use of the composition in the preparation of an enzyme-linked immunoassay kit, particularly in the preparation of a Helicobacter pylori enzyme-linked immunosorbent assay kit.
  • kits for detecting a Helicobacter pylori antibody spectrum comprising the following components:
  • a protein chip coated with a Helicobacter pylori virulence protein antigen an enzyme-labeled antibody diluted with an enzyme standard dilution, a sample diluent, a washing solution, and a color developing solution; wherein the protein chip is coated with a Helicobacter pylori virulence protein
  • the antigen is then blocked with a blocking solution containing BSA, betaine, mannitol, sodium azide, disodium hydrogen phosphate and citric acid, the enzyme standard dilution containing Tris, citric acid, BSA, gum arabic, beets Base and Proclin 300, the blocking solution and the enzyme standard dilution are the same as the previous composition scheme.
  • the Helicobacter pylori virulence protein antigen is selected from the group consisting of CagA, VacA, UreA, UreB, HSP60, gGT, FliD, HtrA, HpaA, CtkA, NapA, HP231, JHP940, Omp, HcpC, Catalase, TonB Or two or more; in a specific embodiment of the present invention, the Helicobacter pylori virulence protein antigens are CagA, VacA, UreA, UreB, HSP60, gGT, FliD, HtrA, HpaA, CtkA, NapA, HP231, JHP940, Omp, HcpC, Catalase and TonB.
  • the Helicobacter pylori virulence protein antigen contains PEG (eg PEG4000), Proclin300 and 2-hydroxy- ⁇ .
  • the cyclodextrin buffer is coated with an antigen coating buffer; preferably, the buffer is selected from the group consisting of CB buffer of pH 9.6, Tris buffer of pH 8.5, and PBS buffer of pH 7.4-7.6.
  • the antigen coating buffer may be specifically selected from a CB buffer of pH 9.6 (containing 5% PEG, 0.05% Proclin 300, and 0.02% 2-hydroxy- ⁇ - Cyclodextrin), pH 8.5 Tris buffer (5% PEG, 0.05% Proclin300, and 0.02% 2-hydroxy- ⁇ -cyclodextrin) and pH 7.4-7.6 in PBS buffer (including 6% PEG, 0.05% Proclin 300, and 0.02% 2-hydroxy- ⁇ -cyclodextrin; more specifically, during the coating process:
  • UreA, UreB, HSP60, FliD, gGT were diluted to a working concentration with a pH 8.5 Tris buffer (5% PEG, 0.05% Proclin 300, and 0.02% 2-hydroxy- ⁇ -cyclodextrin). ;
  • HpaA, HP231, HcpC, JHP940, TonB were diluted to a working concentration with PBS buffer (containing 6% PEG, 0.05% Proclin300, and 0.02% 2-hydroxy- ⁇ -cyclodextrin) in pH 7.4-7.6.
  • the protein chip in the kit of the present invention further comprises one or more of a negative control point, a positive control point, a sample control point, an enzyme label control point, a reference curve point, and a position reference point; Specifically, there is at least one negative property control point (NC) and one positive nature control point (PC); at least one sample point control point (SC) and one enzyme standard control point (EC); at least three reference curve points (S1-S3) and a position reference point (Loc) coated by the chip itself.
  • NC negative property control point
  • PC positive nature control point
  • SC sample point control point
  • EC enzyme standard control point
  • S1-S3 enzyme standard control point
  • the protein chip of the present invention further comprises a negative property control point (NC) and a positive nature control point (PC); a sample point quality control point (SC) and an enzyme standard control point (EC) 3 reference curve points (S1-S3) and a position reference point (Loc) coated by the chip itself.
  • NC negative property control point
  • PC positive nature control point
  • SC sample point quality control point
  • EC enzyme standard control point
  • S1-S3 position reference point coated by the chip itself.
  • the positive control point can be human IgG, and the corresponding enzyme-labeled antibody is an anti-human IgG enzyme label.
  • the positive control point can also be a BNP coated with BSA, and the corresponding enzyme-labeled antibody is a mixture of an anti-human IgG enzyme label and an anti-BNP enzyme label.
  • the negative control point may be a small concentration of human IgG lower than the reaction signal value, or may be replaced by other unrelated proteins; the sample control point may be goat anti-human IgG or other anti-human IgG; The point may be human IgG, or other anti-rabbit antibody, such as a goat anti-rabbit IgG antibody.
  • the reference curve points are low, medium and high concentrations of human IgG in the specific implementation process.
  • the position reference point of the protein chip itself is a DMSO solution containing 0.2% of 2,9-dimethylquinacridone, or an oil-soluble dye containing any color other than blue, and is compatible with an organic solvent. A certain proportion of the ratio, forming an organic colorant, mainly to locate the array on the protein chip.
  • the enzyme label in the enzyme-labeled antibody of the present invention may be selected from conventional enzymes and corresponding color developing solutions such as horseradish peroxidase and TMB color developer.
  • the invention also provides a method for preparing the kit, which comprises:
  • the Helicobacter pylori virulence protein antigen is coated on a protein chip, washed after coating, and then blocked by a blocking solution to obtain a protein chip coated with a Helicobacter pylori virulence protein antigen, the blocking solution containing BSA, beet Alkali, mannitol, sodium azide, disodium hydrogen phosphate, and citric acid;
  • the Helicobacter pylori antibody spectrum detection kit of the present invention can increase the sensitivity (99.5%) and specificity (100%) of HP detection, and is highly consistent with the results of immunohistochemistry.
  • the present invention starts from the blocking solution and the enzyme standard dilution of the enzyme-linked immunoassay kit, and the enzyme-linked immunoassay kit can maintain the stability of the detection for a long time by selecting appropriate components.
  • the H. pylori detection kit prepared by the composition can also have higher sensitivity and specificity for different subtypes of Helicobacter pylori typing on the basis of better stability.
  • the invention discloses a composition for an enzyme-linked immunoassay kit and a Helicobacter pylori antibody spectrum detecting kit and a preparation method thereof, 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 compositions, kits and applications of the present invention have been described in terms of preferred embodiments, and it will be apparent to those skilled in the art that the compositions, kits and applications described herein can be modified without departing from the scope, spirit and scope of the invention. The invention may be implemented and applied by appropriate modifications and combinations.
  • Example 1 Preparation of the kit for detecting Helicobacter pylori antibody spectrum of the present invention
  • PC, NC, S1, S2, S3, and EC in the protein chip array were coated with human IgG at 2 ug/ml, 0.01 ug/ml, 0.5 ug/ml, 2 ug/ml, and 4 ug/ml, respectively.
  • the CB buffer (containing 5% PEG, 0.05% Proclin 300, and 0.02% 2-hydroxy- ⁇ -cyclodextrin) was diluted.
  • the SC point used 2ug/ml goat anti-human IgG antibody, and the dilution buffer was PH9.6 CB buffer (containing 5% PEG, 0.05% Proclin300, and 0.02% 2-hydroxy- ⁇ -cyclodextrin). fine).
  • the Loc point used was a DMSO solution containing 0.2% 2,9-dimethylquinacridone.
  • Catalase dilution buffer is pH 9.6 CB buffer (5% PEG, 0.05% Proclin300, and 0.02% 2-hydroxy- ⁇ -cyclodextrin) ), the final concentrations were 6 ug/ml, 8 ug/ml, 15 ug/ml, 10 ug/ml, 20 ug/ml, 12 ug/ml, 30 ug/ml, respectively.
  • the dilutions of UreA, UreB, HSP60, FliD, and gGT are pH 8.5 Tris buffer (5% PEG, 0.05% Proclin300, and 0.02% 2-hydroxy- ⁇ -cyclodextrin), respectively. It is 10 ug/ml, 10 ug/ml, 15 ug/ml, 12 ug/ml, and 80 ug/ml.
  • the dilution buffers of HpaA, HP231, HcpC, JHP940 and TonB are PBS buffer (containing 6% PEG, 0.05% Proclin300, and 0.02% 2-hydroxy- ⁇ -cyclodextrin) at pH 7.4-7.6.
  • the final concentrations were 15 ug/ml, 30 ug/ml, 15 ug/ml, 60 ug/ml, and 40 ug/ml, respectively.
  • the diluted proteins were separately filtered through a 0.22 um filter, and then coated with an array using a BioDot precision spotter. After all the arrays were spotted, the chip was placed at 2-8 ° C and overnight coated for 24-30 h.
  • the array of protein chips can be referred to the array presented in the following table, or can be adjusted according to actual needs, without limitation:
  • the coated chips were removed and washed 3 times with PBST washing solution of pH 7.4, then 150 ul of blocking solution (0.9% BSA, 1% betaine, 1% mannitol, sodium azide, disodium hydrogen phosphate) was added to each well. And 2% citric acid, pH 7.4), sealed at room temperature for 1h, then patted dry, below 15% humidity, room temperature, dried for 4h, then sealed, stored at 2-8 ° C, obtained Helicobacter pylori virulence Protein chip for protein antigens.
  • blocking solution 0.9% BSA, 1% betaine, 1% mannitol, sodium azide, disodium hydrogen phosphate
  • Enzyme dilution containing 0.1 M Tris, 0.05 M citric acid, 2.5% BSA, 2% gum arabic, 1% betaine and 0.05% Proclin 300;
  • Enzyme-labeled antibody horseradish peroxidase-labeled rabbit anti-human IgG antibody
  • the horseradish peroxidase-labeled rabbit anti-human IgG antibody was diluted to 4K times (enzyme-labeled antibody concentration) with an enzyme standard dilution.
  • Color developing solution Settling type TMB.
  • the above-mentioned Helicobacter pylori antibody spectrum detecting kit comprising the Helicobacter pylori virulence protein antigen protein chip, the enzyme standard antibody diluted with the enzyme standard dilution solution, and the color developing solution, and the sample diluent and the 10-fold concentrated washing liquid .
  • the sensitivity of the chip kit of the present invention to HP infection detection can reach 99.49%, and the specificity (negative correct rate) is 100%, which is highly consistent with the detection results of immunohistochemistry.
  • Example 3 Stability test of the kit of the present invention
  • Control kit prepared according to the method of Example 1, except that the blocking solution and the enzyme standard dilution were both conventional 0.01 M PBS (pH 7.4) + 10% BSA;
  • Test kit the kit of Example 1;
  • Detection method the two kits were placed at room temperature (18-28 ° C) and low temperature (2-8 ° C) for a period of time, and then using the same serum according to the detection method of Example 1, statistical instrument signal values, the results see Table 3-6.
  • kits of the present invention respectively detect the detection signal values at 0, 6, 12, 18, and 24 months at a low temperature, and simultaneously count the ratios of the respective signal values, and the results show that 24 cells are placed.
  • the detection signal value is almost over 90% compared with the detection signal values placed at 0, 6, 12, and 18 months, which proves that the kit of the present invention is placed under low temperature conditions for 24 months. High detection stability.
  • the kit of the present invention detects the detection signal values of 0, 3, 6, 9, and 12 months at normal temperature, and simultaneously counts the ratio of each signal value, and the result shows that 12 are placed.
  • the detection signal value is almost 90% or more compared with the detection signal values placed at 0, 3, 6, and 9 months, which proves that the kit of the present invention is left at room temperature for 12 months. High detection stability.
  • the kit of the present invention and the control kit respectively detect the detection signal values placed at low temperature for 6, 12, 18, and 24 months, and simultaneously count the signal values of the two kits at the same time.
  • the ratio of the results showed that the 18-month control kit showed a significant decrease in the detection signal value.
  • the stability of the control kit is not as good as the conclusion of the kit of the present invention. The only difference is the blocking solution and the enzyme standard dilution.
  • the kit of the present invention and the control kit respectively detect the detection signal values placed at normal temperature for 3, 6, 9, and 12 months, and simultaneously count the signal values of the two kits at the same time.
  • the ratio shows that the control kit placed for 6 months has a significant decrease in the detection signal value.
  • the stability of the control kit is not as good as the conclusion of the kit of the present invention. The only difference is the blocking solution and the enzyme standard dilution.

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Abstract

一种用于酶联免疫试剂盒的组合物以及幽门螺旋杆菌抗体谱检测试剂盒及其制备方法，其中组合物包括封闭液和酶标稳定稀释液；封闭液含有BSA、甜菜碱、甘露醇、叠氮钠、磷酸氢二钠和柠檬酸，酶标稀释液含有Tris、柠檬酸、BSA、阿拉伯树胶、甜菜碱和Proclin300。通过选择封闭液和酶标稀释适宜成分，使得酶联免疫试剂盒能够较长时间保持检测的稳定性，而且该组合物制备的幽门螺旋杆菌检测试剂盒能够对不同亚型的幽门螺旋杆菌分型，具备较高的敏感性和特异性。

Description

一种用于酶联免疫试剂盒的组合物以及幽门螺旋杆菌抗体谱检测试剂盒及其制备方法
本申请要求于2017年07月11日提交中国专利局、申请号为201710561754.8、发明名称为“一种用于酶联免疫试剂盒的组合物以及幽门螺旋杆菌抗体谱检测试剂盒及其制备方法”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
本发明涉及酶联免疫技术领域，具体涉及一种用于酶联免疫试剂盒的组合物以及幽门螺旋杆菌抗体谱检测试剂盒及其制备方法。
背景技术
幽门螺杆菌(Helicobacter pylori,H.pylori)是一种定植于胃黏膜的病原菌，世界范围内约有高达50-70％的感染率。该菌可以引发多种消化道疾病，而且可能与某些消化道以外的疾病有着某种联系，如贫血、消化不良和一些自身免疫疾病。
H.pylori致病力和致癌性取决于其毒力因子，目前已知的毒力因子主要有细胞毒素相关蛋白A(Cytotoxin associated protein A,CagA)、空泡毒素A(Vacuolating toxinA，VacA)、尿素酶A(UreaseA，UreA)、尿素酶B(UreaseB,UreB)、热休克蛋白60(Heat Shock Protein 60,HSP60)、γ-谷氨酰转肽酶(γ-glutamyl transpeptidase,gGT)、鞭毛钩相关蛋白(Flagellar hook-associated protein 2,FliD)、高温热激需求蛋白(High temperature requirement A,HtrA)、幽门螺杆菌鞭毛粘附素(helicobacterpylori adhesin,HpaA)、细胞转运蛋白激酶(Cell translocating kinase，CtkA)、中性白细胞激活蛋白(Neutrophil-activating protein,NapA)、假设蛋白(HP231)，胃癌相关因子(JHP940)，外膜蛋白(Omp)，富含半胱氨酸蛋白C(HcpC)，过氧化氢酶(Catalase)，依赖铁的载体转运蛋白(TonB Protein)。
但并非所有H.pylori都存在上述毒力因子，而不同H.pylori菌株所具有的不同的毒力因子及数量也决定了菌株本身的毒力和致病力及致癌风 险，已有的研究结论表明CagA、VacA和GroEL(HSP60)为识别患者感染高风险H.pylori菌株的潜在标志物，与胃癌的发生有关，能够作为一项H.pylori胃癌发生风险分层指标。携带有不同毒力因子的H.pylori菌株感染导致机体产生针对不同的毒力因子的相应抗体，因此分析感染者血清中的抗体构成及滴度，有助于反映感染者所感染的H.pylori菌株所具有的毒力因子种类和数量，从而预测H.pylori的毒力和致病力以及临床后果，选择适宜的治疗方案，体现精准医疗的精神，降低治疗风险及费用。
针对幽门螺杆菌的诊断已经有许多的临床检测方法，包括侵入性和非侵入性。但无论是哪一种方法，都只能诊断幽门螺杆菌感染的存在与否，对于H.pylori的分类、毒力判断、临床后果预测以及治疗方案的制定都无法提供进一步的帮助，而临床迫切需要一种方便、快捷、准确、敏感的检测方法不仅可以确定H.pylori感染，还可以判断H.pylori感染的类型、毒力强弱以及致病、致癌风险，从而更有效和精准地指导治疗和进行预后判断。
ELISA(酶联免疫吸附试验)，指将可溶性的抗原或抗体结合到聚苯乙烯等固相载体上，利用抗原抗体结合专一性进行免疫反应的定性和定量检测方法，是酶免疫测定技术中应用最广的技术。中国专利CN102721815A提供了一种幽门螺旋杆菌毒力蛋白抗体检测试剂盒，其基于ELISA原理，以6种幽门螺旋杆菌毒力蛋白作为抗原对患者血清进行检测，以此判断菌株毒力强弱和实现半定量检测。但是，该试剂盒的稳定性并不好，这也是所有类型酶联免疫试剂盒所面临的共同问题，此外，该试剂盒仍然无法区分各亚型的幽门螺旋杆菌，检测特异性和敏感性也不尽如人意。
发明内容
有鉴于此，本发明的目的在于提供一种用于酶联免疫试剂盒的组合物，使得所述组合物用于制备酶联免疫试剂盒时能够显著提高试剂盒在低温和室温下的稳定性，保存时间延长；
本发明的另外一个目的在于提供上述组合物在制备酶联免疫试剂盒中的应用，特别是用于检测幽门螺旋杆菌的相关试剂盒；
本发明的另外一个目的在于提供一种包含上述组合物的幽门螺旋杆菌抗体谱检测试剂盒及其制备方法，使得所述试剂盒在低温和室温下具有较长时间的稳定性，同时检测结果具有较高的特异性和敏感性。
为了实现上述目的，本发明提供如下技术方案：
一种用于酶联免疫试剂盒的组合物，包括封闭液和酶标稀释液；所述封闭液含有BSA、甜菜碱、甘露醇、叠氮钠、磷酸氢二钠和柠檬酸，所述酶标稀释液含有Tris、柠檬酸、BSA、阿拉伯树胶、甜菜碱和Proclin300。
针对现有酶联免疫试剂盒稳定较差、保存时间较短的缺陷，本发明意外发现了从制备试剂盒的封闭液和酶标稀释液(用于稀释酶标抗原或抗体用)入手，通过选择合适的组分完善两者的组成，能够显著提高酶联免疫试剂盒的稳定性和保存时间。
作为优选，所述封闭液含有0.6％-1％BSA，0.8％-1.5％的甜菜碱、0.8％-1.5％甘露醇、0.02％叠氮钠、0.01M磷酸氢二钠和1.5％-2.5％柠檬酸，pH值为7.4，余量为水，所述百分比为质量百分比(w/v)；在本发明具体实施方式中，所述封闭液含有0.9％BSA，1％的甜菜碱、1％甘露醇、0.02％叠氮钠、0.01M磷酸氢二钠和2％柠檬酸，pH值为7.4，余量为超纯水。
作为优选，所述酶标稀释液含有0.1M的Tris、0.05M的柠檬酸、2％-3％的BSA、1.8％-2.4％的阿拉伯树胶、0.8％-1.5％的甜菜碱以及0.05％的Proclin300，余量为水，所属百分比中除Proclin300是体积百分比之外，其余都是质量百分比(w/v)；在本发明具体实施方式中，所述酶标稀释液含有0.1M的Tris、0.05M的柠檬酸、2.5％的BSA、2％的阿拉伯树胶、1％的甜菜碱以及0.05％的Proclin300。
本发明利用上述组合物进行幽门螺旋杆菌酶联免疫试剂盒的制备，与采用常规的封闭液和酶标稀释液幽门螺旋杆菌酶联免疫试剂盒相比，在低温(2-8℃)下，本发明试剂盒在放置24个月后仍然能够保持制备完成时的稳定性，而对照的试剂盒在放置18个月后就已经出现很大程度的 不稳定性；在室温(18-28℃)下，本发明试剂盒在放置12个月后仍然能够保持制备完成时的稳定性，而对照的试剂盒在放置6个月后就已经出现很大程度的不稳定性。
基于上述优异的技术效果，本发明提出了所述组合物在制备酶联免疫试剂盒中的应用，特别是在制备幽门螺杆菌酶联免疫检测试剂盒中的应用。
同时，本发明还提供一种幽门螺旋杆菌抗体谱检测试剂盒，包括以下组分：
包被有幽门螺旋杆菌毒力蛋白抗原的蛋白芯片、用酶标稀释液稀释的酶标抗体、样品稀释液、洗涤液和显色液；其中，所述蛋白芯片包被幽门螺旋杆菌毒力蛋白抗原后采用封闭液封闭，所述封闭液含有BSA、甜菜碱、甘露醇、叠氮钠、磷酸氢二钠和柠檬酸，所述酶标稀释液含有Tris、柠檬酸、BSA、阿拉伯树胶、甜菜碱和Proclin300，所述封闭液和酶标稀释液与前述组合物方案相同。
作为优选，所述幽门螺旋杆菌毒力蛋白抗原选自CagA、VacA、UreA、UreB、HSP60、gGT、FliD、HtrA、HpaA、CtkA、NapA、HP231，JHP940，Omp，HcpC，Catalase，TonB中的一种或两种以上；在本发明具体实施方式中，所述幽门螺旋杆菌毒力蛋白抗原为CagA、VacA、UreA、UreB、HSP60、gGT、FliD、HtrA、HpaA、CtkA、NapA、HP231，JHP940，Omp，HcpC，Catalase和TonB。
在包被中，为了使得包被更稳定、抗原包被点更规则、更圆，CV更小，所述幽门螺旋杆菌毒力蛋白抗原采用含有PEG(例如PEG4000)、Proclin300以及2-羟基-β-环糊精的缓冲液为抗原包被缓冲液进行包被；作为优选，所述缓冲液选自PH9.6的CB缓冲液、PH8.5的Tris缓冲液和PH7.4-7.6的PBS缓冲液；在本发明具体实施方式中，所述抗原包被缓冲液可具体选自PH9.6的CB缓冲液(含有5％的PEG、0.05％的Proclin300，以及0.02％的2-羟基-β-环糊精)、PH8.5的Tris缓冲液(含5％的PEG、0.05％的Proclin300，以及0.02％的2-羟基-β-环糊精)和PH7.4-7.6的PBS缓冲液(含6％的PEG、0.05％的Proclin300，以及0.02％的2-羟基-β-环 糊精)；更为具体地，在包被过程中：
CagA、VacA、HtrA、CtkA、NapA、Omp、Catalase采用PH9.6的CB缓冲液(含有5％的PEG、0.05％的Proclin300，以及0.02％的2-羟基-β-环糊精)稀释至工作浓度进行包被；
UreA、UreB、HSP60、FliD、gGT采用PH8.5的Tris缓冲液(含5％的PEG、0.05％的Proclin300，以及0.02％的2-羟基-β-环糊精)稀释至工作浓度进行包被；
HpaA、HP231、HcpC、JHP940、TonB采用PH7.4-7.6的PBS缓冲液(含6％的PEG、0.05％的Proclin300，以及0.02％的2-羟基-β-环糊精)稀释至工作浓度进行包被；
此外，本发明试剂盒中的蛋白芯片还包括阴性质控点、阳性质控点、样品质控点、酶标质控点、参考曲线点以及位置参考点中的一个或两个以上；更为具体地，至少有一个阴性质控点(NC)和一个阳性质控点(PC)；至少一个样品点质控点(SC)和一个酶标质控点(EC)；至少3个参考曲线点(S1-S3)以及一个芯片本身包被的位置参考点(Loc)。
在具体实施方式中，本发明蛋白芯片上还包含一个阴性质控点(NC)和一个阳性质控点(PC)；一个样品点质控点(SC)和一个酶标质控点(EC)；3个参考曲线点(S1-S3)以及一个芯片本身包被的位置参考点(Loc)。
其中，阳性质控点可以是人IgG，则对应使用的酶标抗体就是抗人IgG的酶标。阳性质控点也可以是包被BSA偶联的BNP，则对应使用的酶标抗体就是抗人IgG的酶标以及抗BNP的酶标的混合液。
而阴性质控点可以是低于反应信号值的微量浓度的人IgG，或采用其他的无关蛋白来替代；样品质控点可以是羊抗人的IgG或其他的抗人IgG；酶标质控点可以是人IgG，或其他的抗兔的抗体，例如羊抗兔IgG抗体。所述参考曲线点在具体实施过程中是低、中、高三种浓度的人IgG。
蛋白芯片本身的位置参考点是含有0.2％的2,9-二甲基喹吖啶酮的DMSO溶液，或是含有任何除蓝色之外其他颜色的油溶性染剂，与有机溶剂配伍，按一定比例配比，形成有机着色剂，主要对蛋白芯片上阵列 的起定位作用。
本发明所述酶标抗体中酶标记物可选择常规的酶以及对应的显色液，如辣根过氧化物酶和TMB显色剂。
本发明还对应提供了所述试剂盒的制备方法，包括：
将幽门螺旋杆菌毒力蛋白抗原在蛋白芯片上进行包被，包被后洗涤，然后加入封闭液封闭，获得包被有幽门螺旋杆菌毒力蛋白抗原的蛋白芯片，所述封闭液含有BSA、甜菜碱、甘露醇、叠氮钠、磷酸氢二钠和柠檬酸；
配制含有Tris、柠檬酸、BSA、阿拉伯树胶、甜菜碱和Proclin300的酶标稀释液并稀释酶标抗体，获得用酶标稀释液稀释的酶标抗体，然后配制样品稀释液、洗涤液和显色液，获得幽门螺旋杆菌抗体谱检测试剂盒。
采用本发明幽门螺旋杆菌抗体谱检测试剂盒能够提高HP检测的敏感性(99.5％)和特异性(100％)，与免疫组化结果高度一致。
由以上技术方案可知，本发明从酶联免疫试剂盒的封闭液和酶标稀释液入手，通过选择适宜成分，使得酶联免疫试剂盒能够较长时间保持检测的稳定性。同时，以所述组合物制备的幽门螺旋杆菌检测试剂盒在具有较佳稳定性的基础上，还能够对不同亚型的幽门螺旋杆菌分型，具备较高的敏感性和特异性。
具体实施方式
本发明公开了一种用于酶联免疫试剂盒的组合物以及幽门螺旋杆菌抗体谱检测试剂盒及其制备方法，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。本发明所述组合物、试剂盒和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组合物、试剂盒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。
以下就本发明所提供的一种用于酶联免疫试剂盒的组合物以及幽门螺旋杆菌抗体谱检测试剂盒及其制备方法做进一步说明。
实施例1：本发明所述幽门螺旋杆菌抗体谱检测试剂盒的制备
1、幽门螺旋杆菌毒力蛋白抗原和相关蛋白的包被
蛋白芯片阵列中的PC、NC、S1、S2、S3、EC分别包被的是2ug/ml、0.01ug/ml、0.5ug/ml、2ug/ml、4ug/ml的人IgG，可用PH9.6的CB缓冲液(含5％的PEG、0.05％的Proclin300，以及0.02％的2-羟基-β-环糊精)进行稀释。
SC点使用的是2ug/ml的羊抗人IgG抗体，稀释缓冲液为PH9.6的CB缓冲液(含5％的PEG、0.05％的Proclin300，以及0.02％的2-羟基-β-环糊精)。
Loc点使用的是含有0.2％的2,9-二甲基喹吖啶酮的DMSO溶液。
CagA、VacA、HtrA、CtkA、NapA、Omp、Catalase的稀释缓冲液为PH9.6的CB缓冲液(含5％的PEG、0.05％的Proclin300，以及0.02％的2-羟基-β-环糊精)，终浓度分别为6ug/ml、8ug/ml、15ug/ml、10ug/ml、20ug/ml、12ug/ml、30ug/ml。
UreA、UreB、HSP60、FliD、gGT的稀释液为PH8.5的Tris缓冲液(含5％的PEG、0.05％的Proclin300，以及0.02％的2-羟基-β-环糊精)，终浓度分别为10ug/ml、10ug/ml、15ug/ml、12ug/ml、80ug/ml。
HpaA、HP231、HcpC、JHP940、TonB的稀释缓冲液为PH7.4-7.6的PBS缓冲液(含6％的PEG、0.05％的Proclin300，以及0.02％的2-羟基-β-环糊精),终浓度分别为15ug/ml、30ug/ml、15ug/ml、60ug/ml、40ug/ml。
将稀释好的蛋白分别用0.22um的滤膜过滤，然后用BioDot精密点样仪进行阵列的包被。全部阵列完成点样之后，将芯片置于2-8℃，过夜包被24-30h。蛋白芯片阵列可参照如下表呈现的阵列，也可根据实际需要调整，不做限制：
表1蛋白芯片阵列
PC CagA gGT NapA Catalase
NC VacA FliD HP231 TonB
S1 UreA HtrA JHP940 SC
S2 UreB HpaA Omp EC
S3 HSP60 CtkA HcpC Loc
2、封闭
取出包被的芯片，用PH7.4的PBST洗涤液清洗3次，然后每孔加入150ul的封闭液(0.9％BSA，1％的甜菜碱、1％甘露醇、叠氮钠、磷酸氢二钠和2％柠檬酸，pH值为7.4)，室温封闭1h，然后拍干，于湿度15％以下，室温放置，干燥4h，后密封、2-8℃保存，获得包被有幽门螺旋杆菌毒力蛋白抗原的蛋白芯片。
3、配制酶标稀释液、酶标抗体、显色液、样品稀释液和浓缩洗涤液
酶标稀释液：含有0.1M的Tris、0.05M的柠檬酸、2.5％的BSA、2％的阿拉伯树胶、1％的甜菜碱以及0.05％的Proclin300；
酶标抗体：辣根过氧化物酶标记的兔抗人IgG抗体；
使用时，用酶标稀释液将辣根过氧化物酶标记的兔抗人IgG抗体稀释至4K倍(酶标抗体浓度)。
显色液：沉降型TMB。
样品稀释液：0.02M Tris，0.15M NaCl，0.05％Tween20，0.01％酪蛋白，pH7.4。
10倍浓缩洗涤液：0.2M Tris，1.5M NaCl，0.5％Tween20，pH7.4。
上述包被有幽门螺旋杆菌毒力蛋白抗原的蛋白芯片、酶标稀释液稀释的酶标抗体和显色液、以及样品稀释液和10倍浓缩洗涤液组成本发明幽门螺旋杆菌抗体谱检测试剂盒。
4、检测方法
(1)取出蛋白芯片，平衡至室温；
(2)加样：将阴性和阳性对照血清、以及用样品稀释液稀释了101倍的待测样品，每孔100uL加入待测芯片孔中反应。
(3)温育：室温静置反应30min。加300uL洗涤液(用超纯水稀释10倍后使用)，洗涤3次，每次静置1min。
(4)加酶标抗体：每孔加入50uL酶标抗体。
(5)温育：室温静置反应30min。加300uL洗涤液，洗涤3次，每次静置1min。
(6)显色：每孔加入TMB显色剂50uL，室温静置，避光反应30min。
(7)测定：30min内，用检测仪读取并计算每个反应孔对应抗体的信号值；其中，信号检测体系也可以是化学发光的方式来实现，可用化学发光底物，如鲁米诺等，然后通过荧光检测装置来进行结果的读取。
实施例2：敏感性和特异性检测
选取263例临床血清样本，其中阴性样本68例，阳性样本195例，所有样本均通过免疫组化实验诊断为幽门螺杆菌感染阳性、阴性患者的血清，具体的实验结果和数据如下表：
表2
Figure PCTCN2018093060-appb-000001
由表2可知，本发明的芯片试剂盒对HP感染检测的敏感度(阳性正确率)可达到99.49％，特异性(阴性正确率)100％，与免疫组化的检测结果高度一致。
实施例3：本发明所述试剂盒稳定性检测
对照试剂盒：按照实施例1的方法进行制备，区别在于封闭液和酶标稀释液均采用常规的0.01M PBS(PH7.4)+10％BSA；
试验试剂盒：实施例1试剂盒；
检测方法：将两种试剂盒分别置于室温(18-28℃)和低温(2-8℃)下放置一段时间，然后采用相同血清按照实施例1检测方法检测，统计仪器信号值，结果见表3-6。
1、实施例1试剂盒低温下的稳定性数据
表3
Figure PCTCN2018093060-appb-000002
Figure PCTCN2018093060-appb-000003
Figure PCTCN2018093060-appb-000004
由表3可以看出，本发明试剂盒分别检测了在低温下放置0、6、12、18、24个月的检测信号值，同时统计了各个信号值的比值，结果显示，放置了24个月的试剂盒，其检测信号值与放置0、6、12、18个月的检测信号值相比，几乎均处于90％以上，证明本发明所述试剂盒在低温条件下放置24个月仍然具备较高的检测稳定性。
2、实施例1试剂盒室温下的稳定性数据
表4
Figure PCTCN2018093060-appb-000005
Figure PCTCN2018093060-appb-000006
Figure PCTCN2018093060-appb-000007
由表4可以看出，本发明试剂盒分别检测了在常温下放置0、3、6、9、12个月的检测信号值，同时统计了各个信号值的比值，结果显示，放置了12个月的试剂盒，其检测信号值与放置0、3、6、9个月的检测信号值相比，几乎均处于90％以上，证明本发明所述试剂盒在常温条件下放置12个月仍然具备较高的检测稳定性。
3、实施例1试剂盒和对照试剂盒低温下的稳定性数据对比
表5
Figure PCTCN2018093060-appb-000008
Figure PCTCN2018093060-appb-000009
由表5可以看出，本发明试剂盒和对照试剂盒分别检测了在低温下放置6、12、18、24个月的检测信号值，同时统计了相同时间下，两个试剂盒的信号值的比值，结果显示，放置了18个月的对照试剂盒，其检测信号值开始出现显著的下降，结合表3数据可以明显得出对照试剂盒的稳定性不如本发明试剂盒的结论，而两者的差别仅在于封闭液和酶标稀释液。
4、实施例1试剂盒和对照试剂盒常温下的稳定性数据对比
表6
Figure PCTCN2018093060-appb-000010
Figure PCTCN2018093060-appb-000011
由表6可以看出，本发明试剂盒和对照试剂盒分别检测了在常温下放置3、6、9、12个月的检测信号值，同时统计了相同时间下，两个试剂盒的信号值的比值，结果显示，放置了6个月的对照试剂盒，其检测信号值开始出现显著的下降，结合表4数据可以明显得出对照试剂盒的稳定性不如本发明试剂盒的结论，而两者的差别仅在于封闭液和酶标稀释液。
以上所述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 (12)

  1. 一种用于酶联免疫试剂盒的组合物，其特征在于，包括封闭液和酶标稀释液；所述封闭液含有BSA、甜菜碱、甘露醇、叠氮钠、磷酸氢二钠和柠檬酸，所述酶标稀释液含有Tris、柠檬酸、BSA、阿拉伯树胶、甜菜碱和Proclin300。
  2. 根据权利要求1所述组合物，其特征在于，所述封闭液含有0.6％-1％BSA，0.8％-1.5％的甜菜碱、0.8％-1.5％甘露醇、0.02％叠氮钠、0.01M磷酸氢二钠和1.5％-2.5％柠檬酸，pH值为7.4，余量为水。
  3. 根据权利要求1所述组合物，其特征在于，所述酶标稀释液含有0.1M的Tris、0.05M的柠檬酸、2％-3％的BSA、1.8％-2.4％的阿拉伯树胶、0.8％-1.5％的甜菜碱以及0.05％的Proclin300，余量为水。
  4. 权利要求1-3任意一项所述组合物在制备酶联免疫试剂盒中的应用。
  5. 根据权利要求4所述应用，其特征在于，所述酶联免疫试剂盒为幽门螺杆菌酶联免疫检测试剂盒。
  6. 一种幽门螺旋杆菌抗体谱检测试剂盒，其特征在于，包括以下组分：
    包被有幽门螺旋杆菌毒力蛋白抗原的蛋白芯片、用酶标稀释液稀释的酶标抗体、样品稀释液、洗涤液和显色液；其中，所述蛋白芯片包被幽门螺旋杆菌毒力蛋白抗原后采用封闭液封闭，所述封闭液含有BSA、甜菜碱、甘露醇、叠氮钠、磷酸氢二钠和柠檬酸，所述酶标稀释液含有Tris、柠檬酸、BSA、阿拉伯树胶、甜菜碱和Proclin300。
  7. 根据权利要求6所述试剂盒，其特征在于，所述封闭液含有0.6％-1％BSA，0.8％-1.5％的甜菜碱、0.8％-1.5％甘露醇、0.02％叠氮钠、0.01M磷酸氢二钠和1.5％-2.5％柠檬酸，pH值为7.4，余量为水。
  8. 根据权利要求6所述试剂盒，其特征在于，所述酶标稀释液含有0.1M的Tris、0.05M的柠檬酸、2％-3％的BSA、1.8％-2.4％的阿拉伯树胶、0.8％-1.5％的甜菜碱以及0.05％的Proclin300，余量为水。
  9. 根据权利要求6所述试剂盒，其特征在于，所述幽门螺旋杆菌毒力蛋白抗原选自CagA、VacA、UreA、UreB、HSP60、gGT、FliD、HtrA、HpaA、CtkA、NapA、HP231，JHP940，Omp，HcpC，Catalase，TonB中的一种或两种以上。
  10. 根据权利要求6所述试剂盒，其特征在于，所述幽门螺旋杆菌毒力蛋白抗原采用含有PEG、Proclin300以及2-羟基-β-环糊精的缓冲液为抗原包被缓冲液进行包被。
  11. 根据权利要求2-10任意一项所述试剂盒，其特征在于，所述蛋白芯片还包括阴性质控点、阳性质控点、样品质控点、酶标质控点、参考曲线点以及位置参考点中的一个或两个以上。
  12. 权利要求6所述试剂盒的制备方法，其特征在于，包括：
    将幽门螺旋杆菌毒力蛋白抗原在蛋白芯片上进行包被，包被后洗涤，然后加入封闭液封闭，获得包被有幽门螺旋杆菌毒力蛋白抗原的蛋白芯片，所述封闭液含有BSA、甜菜碱、甘露醇、叠氮钠、磷酸氢二钠和柠檬酸；
    配制含有Tris、柠檬酸、BSA、阿拉伯树胶、甜菜碱和Proclin300的酶标稀释液并稀释酶标抗体，获得用酶标稀释液稀释的酶标抗体，然后配制样品稀释液、洗涤液和显色液，获得幽门螺旋杆菌抗体谱检测试剂盒。
PCT/CN2018/093060 2017-07-11 2018-06-27 一种用于酶联免疫试剂盒的组合物以及幽门螺旋杆菌抗体谱检测试剂盒及其制备方法 WO2019011125A1 (zh)

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