WO2019169800A1 - 一种用于酶联免疫试剂盒的组合物以及肿瘤标志物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肿瘤标志物检测试剂盒及其制备方法 Download PDF

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WO2019169800A1
WO2019169800A1 PCT/CN2018/093059 CN2018093059W WO2019169800A1 WO 2019169800 A1 WO2019169800 A1 WO 2019169800A1 CN 2018093059 W CN2018093059 W CN 2018093059W WO 2019169800 A1 WO2019169800 A1 WO 2019169800A1
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Prior art keywords
enzyme
kit
tumor marker
trehalose
antibody
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PCT/CN2018/093059
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English (en)
French (fr)
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张大准
张永顶
马伟民
王洪涛
马新民
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深圳市伯劳特生物制品有限公司
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Publication of WO2019169800A1 publication Critical patent/WO2019169800A1/zh

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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
    • 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
    • G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • 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
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

Definitions

  • the invention relates to the field of enzyme-linked immunoassay, in particular to a composition for an enzyme-linked immunoassay kit, a tumor marker detection kit and a preparation method thereof.
  • Tumors especially malignant tumors (cancers)
  • cancers are a type of frequently-occurring and common disease that poses a serious threat to human health.
  • the incidence and mortality of cancer have been on the rise, and there are problems of late detection and untimely treatment, resulting in higher mortality.
  • early detection and prevention are still effective methods for prevention and treatment of cancer.
  • Tumor markers are substances produced by tumor cells themselves or caused by the body's reaction to tumor cells during the process of tumorigenesis and proliferation. They reflect the presence and growth of tumors, including proteins, hormones, enzymes, Polyamines and oncogenes.
  • AFP alpha fetoprotein
  • CEA carcinoembryonic antigen
  • CA242 cancer antigen 242
  • CA125 cancer antigen
  • CA15-3) cancer antigen 15-3 (CA15-3)
  • cancer antigen 72-4 cancer antigen 50
  • CA50 ferritin
  • SF growth hormone
  • TPSA chorionic gonadotropin beta subunit
  • TPSA total prostate specific antigen
  • FPSA free prostate specific antigen
  • CT squamous cell carcinoma antigen
  • NSE neuron enolase
  • NSE non-small cell lung cancer-associated antigen
  • CYFRA21-1 non-small cell lung cancer-associated antigen
  • Pro-GRP tumor-GRP
  • tumor markers are organ-free and are broad-spectrum markers. For example, it is difficult to measure CEA in the blood of normal adults. 70-90% of patients with colon adenocarcinoma are highly positive in CEA. The positive rate in other malignant tumors is gastric cancer (60-90%) and pancreatic cancer (70-80). %), small intestine adenocarcinoma (60-83%), lung cancer (56-80%), liver cancer (62-75%), breast cancer (40-68%), urinary cancer (31-46%). At the same time, a cancer patient often has abnormalities or obvious elevations of various tumor markers. Therefore, the detection of single indicators generally has the problems of low specificity, low positive rate, and especially low detection rate of early tumors. .
  • Simultaneous detection of multiple tumor markers can significantly improve the detection rate of related tumors, and can also help to determine the type of tumor more effectively, in order to assist in the further diagnosis and determination of treatment options.
  • combined determination of CEA, PGI, PGII, CA19-9, CA72-4 can greatly improve the detection rate of gastric cancer; simultaneous detection of AFP, SF, CA125 is conducive to the judgment of liver cancer; and CEA, SCC, ProGRP, CYFRA21- 1, NSE, CA125, etc. are markers of lung cancer; and so on.
  • the joint detection of multiple indicators of tumor markers has gradually attracted the attention of most scholars, and will also receive more and more attention in the clinic.
  • the combined detection of multiple tumor markers is especially suitable for the anti-cancer screening of healthy or sub-healthy people, which is conducive to the early detection of tumors and provides a means of detection for clinical diagnosis and treatment.
  • the most commonly used detection methods for tumor markers include radioimmunoassay, enzyme-linked immunosorbent assay, chemiluminescence immunoassay, immunoblotting, etc.
  • the commonly used enzyme-linked immunosorbent assay has high sensitivity and specificity. Good, but the detection index is single, can not achieve the purpose of census; radioimmunoassay has the risk of pollution; chemiluminescence immunoassay has short luminescence time, need to strictly control the measurement time defects; immunoblot sensitivity is low, operation is cumbersome, and can not be quantified Determination.
  • CN103869068 which has low cost, sensitivity, accuracy, high throughput, and low sample usage. And can be promoted and scaled in the general laboratory, but the kit needs to be equipped with standard products, and by diluting the standard products by different multiples and then measuring the signal value, to develop a standard curve for the quantification of the sample to be tested, still More cumbersome.
  • patent CN103869068 discloses a high-throughput, high-sensitivity tumor marker antibody chip kit, which is inexpensive, sensitive, accurate, high-throughput, and has a small amount of sample, and can be promoted and scaled in an ordinary laboratory. And so on, but the kit needs to be prepared by standardizing and diluting the standard by different multiples and then measuring the signal value to establish a standard curve for the quantification of the sample to be tested, which is still cumbersome. What is more noteworthy is that the stability of this type of kit is not good at present.
  • 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, in particular, a kit for detecting a tumor marker;
  • Another object of the present invention is to provide a tumor marker detecting kit comprising the above composition and a preparation method thereof, which enable the kit to have a long-term stability at a low temperature and a room temperature, and the detection result is high. 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 fetal bovine serum, trehalose, PBS, Tween 20, PVA and sodium azide; Contains Tris, casein, trehalose, sodium p-hydroxybenzoate, gum arabic, PEG and Proclin300.
  • the present invention unexpectedly finds that starting from the preparation of the kit's blocking solution and the enzyme standard dilution (for diluting the enzyme label), by selecting appropriate The components complement the composition of both, which can significantly improve the stability and storage time of the enzyme-linked immunoassay kit.
  • the blocking solution contains 20%-25% fetal bovine serum, 2%-5% trehalose, 0.01M PBS, 0.02%-0.05% Tween20, 1%-3% PVA, 0.05% sodium azide, pH
  • the value is 7.4, the balance is water, and the percentage is mass percentage (w/v); in a specific embodiment of the invention, 20% fetal bovine serum, 4% trehalose, 0.01 M PBS, 0.05% Tween 20, 1 % PVA and 0.05% sodium azide, pH 7.4, the balance is ultrapure water.
  • the polyvinyl alcohol PVA is preferably polyvinyl alcohol 2W.
  • the enzyme standard dilution contains 0.1 M Tris, 2.5%-5% casein, 2%-5% trehalose, 0.2%-0.5% sodium p-hydroxybenzoate, 0.01%-0.05% gum arabic, 1% - 5% PEG, 0.05% proclin 300, the balance being water.
  • the percentage is the mass percentage (w/v) except Proclin 300 is a volume percentage; in a specific embodiment of the invention, the enzyme standard dilution contains 0.1 M Tris, 2.5% casein, 2% seaweed Sugar, 0.2% sodium p-hydroxybenzoate, 0.01% gum arabic, 1% PEG and 0.05% Proclin 300, the balance being water.
  • the polyethylene glycol is preferably polyethylene glycol 1W.
  • the invention utilizes the above composition to prepare a tumor marker antibody enzyme-linked immunosorbent kit, and compares with a conventional blocking solution and an enzyme standard dilution tumor marker antibody enzyme-linked immunosorbent kit at a low temperature (2-8 ° C)
  • the kit of the present invention was able to maintain a high stability even after 24 months of standing, while the control kit showed a large degree of instability after 18 months of storage; at room temperature (18-28 ° C)
  • the kit of the present invention can maintain the stability at the completion of preparation after being left for 6 months, and can maintain high stability after being placed for 9 months, while the control kit has been placed for 6 months. A large degree of instability occurred and the stability of the test could not be guaranteed after 9 months of placement.
  • the present invention proposes the use of the composition in the preparation of an enzyme-linked immunoassay kit, particularly in the preparation of a tumor marker enzyme-linked immunosorbent assay kit.
  • the present invention also provides a tumor marker detection kit comprising the following components:
  • a protein chip coated with a tumor marker antibody, a biotinylated tumor marker antibody, an enzyme label containing an enzyme-labeled avidin, a sample diluent, a washing solution, and a color developing solution The protein chip is coated with a tumor marker and blocked with a blocking solution containing fetal bovine serum, trehalose, PBS, Tween20, PVA and sodium azide, the enzyme standard dilution containing Tris, casein, seaweed
  • the sugar, sodium p-hydroxybenzoate, gum arabic, PEG and Proclin 300, the blocking solution and the enzyme standard dilution are the same as the previous composition scheme.
  • the enzyme-labeled avidin is horseradish peroxidase-labeled avidin.
  • the avidin is preferably streptavidin.
  • the enzyme standard further comprises an anti-human Ig-HRP, which can be used for a protein chip having a sample control point.
  • the tumor marker is selected from the group consisting of AFP, CEA, CA19-9, CA242, CA125, CA15-3, CA72-4, CA50, SF, HGH, ⁇ -HCG, TPSA, FPSA, CT, SCC, NSE, One or more of CYFRA21-1, Pro-GRP, TSGF, POA, PGI, and PGII. In a particular embodiment of the invention, the invention selects the 22 tumor markers described above.
  • the tumor marker is coated with a tumor marker dilution buffer in CB buffer, PBS buffer or Tris buffer; preferably, the buffer is selected from a CB buffer of pH 9.6, PBS buffer pH 7.4 (preferably 0.01 M) or Tris buffer pH 8.5 (preferably 20 mM), more preferably PEG or PVP, glycerin and Proclin 300 are added to the buffer, while water-soluble cyclodextrin is added. Fine, the coating can be more stable, the tumor markers are more regular, more rounded, and the CV is smaller.
  • the water-soluble cyclodextrin may be Captisol, 2-hydroxy- ⁇ -cyclodextrin or carboxymethyl- ⁇ -cyclodextrin at a concentration of 0.02%; the concentration of PEG is 5%, and the concentration of PVP is 0.5- 0.6%, the concentration of glycerol was 15%, and the concentration of Proclin 300 was 0.05%, except for Proclin 300, which is a percentage by volume, and the rest are mass percentage (w/v).
  • the dilution buffer of CT antibody, SCC antibody, CYFRA21-1 antibody, and TSGF antibody is 20 mM Tris buffer (containing 0.5% PVP, 0.05% Proclin 300, 0.02% Captisol, pH 8.5, And 15% glycerol), the final concentration was 6ug/ml, 10ug/ml, 12ug/ml, 10ug/ml.
  • AFP antibody, CEA antibody, CA19-9 antibody, CA242 antibody, CA125 antibody, CA15-3 antibody, CA72-4 antibody, CA50 antibody, SF antibody, POA antibody dilution buffer is pH 9.6 CB buffer, final concentration They were 5 ug/ml, 6 ug/ml, 8 ug/ml, 8 ug/ml, 6 ug/ml, 6 ug/ml, 5 ug/ml, 6 ug/ml, 10 ug/ml, and 10 ug/ml, respectively.
  • the dilution buffer of HGH antibody, ⁇ -HCG antibody, TPSA antibody, FPSA antibody, NSE antibody, Pro-GRP antibody, PGI antibody, PGII antibody is 0.01 M PBS buffer (containing 0.6% PVP, 0.05). % Proclin300, 0.02% Captisol, and 15% glycerol), final concentrations of 6ug/ml, 6ug/ml, 8ug/ml, 8ug/ml, 10ug/ml, 7ug/ml, 6ug/ml, 6ug/ml .
  • Each of the above tumor marker antibodies is preferably a monoclonal antibody.
  • 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 reference threshold point, an enzyme label control point, a standard curve point, and a position reference point. More specifically, 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 3 standard curves Point (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 standard curves Point
  • Lic position reference 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 may be a certain concentration of biotinylated BSA
  • the negative control point may be a biotinylated BSA lower than a trace concentration reflecting the signal value, or may be replaced by other unrelated proteins
  • the control points can be biotinylated BSA, or other biotinylated extraneous proteins
  • sample point control points (SC) and standard curve points are different concentrations of biotinylated BSA.
  • the position reference point (Loc) of the chip itself is biotinylated BSA, which is mainly for the positioning of the array.
  • the enzyme in the enzyme-labeled avidin of the present invention may be selected from a conventional enzyme and a corresponding color developing solution such as horseradish peroxidase.
  • the color developing liquid used in the present invention is an enhanced chemiluminescent substrate (ECL), and the result of the reaction is read by a fluorescence detecting device or an instrument.
  • ECL enhanced chemiluminescent substrate
  • Other chromogenic substrates such as p-NPP, TMB, and the like can also be used in other embodiments.
  • the invention also provides a method for preparing the kit, which comprises:
  • the tumor marker antibody is coated on a protein chip, washed after coating, and then blocked by adding a blocking solution to obtain a protein chip coated with a tumor marker antibody, which contains fetal bovine serum, trehalose, PBS, Tween20, PVA and sodium azide;
  • Formulate an enzyme-labeled dilution containing Tris, casein, trehalose, sodium p-hydroxybenzoate, acacia, PEG, and Proclin300 and dilute the enzyme label containing the enzyme-labeled avidin to obtain an enzyme-labeled parent diluted with the enzyme-labeled dilution Then, a biotinylated tumor marker antibody is prepared, and a sample diluent, a washing solution, and a color developing solution are prepared to obtain a tumor marker detecting kit.
  • the serum sample is detected by the tumor marker detection kit of the present invention, and the detection rate of the clinical sample of the CEA and FPSA at the lower limit of the reference range can be 100% (compared with the existing commercially available kit), and Compared with the commercially available kits, the correlation between the two can reach R2>0.98 (R>0.99), which indicates that the accuracy and sensitivity of the kit of the present invention meet the expected requirements.
  • 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 tumor marker detection kit prepared by the composition has higher sensitivity, accuracy and precision on the basis of better stability.
  • the invention discloses a composition for an enzyme-linked immunoassay kit and a tumor marker detection 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.
  • composition for an enzyme-linked immunoassay kit and a tumor marker detection kit and a preparation method thereof according to the present invention are further described.
  • Example 1 Preparation of the tumor marker detection kit of the present invention
  • Loc, EC, PC, NC, S1, S2, and S3 in the protein chip array are respectively coated with 2 ug/ml, 2 ug/ml, 2 ug/ml, 0.01 ug/ml, 0.5 ug/ml, 2 ug/ml, 4ug/ml biotinylation (Thermo's The BSA of the Sulfo-NHS-LC-Biotin biotinylation kit can be diluted with C9.6 buffer of pH 9.6 (containing 5% PEG400, 0.05% Proclin 300, and 0.02% Captisol).
  • the sample control point was 1.5 ug/ml anti-human IgG and the dilution buffer was pH 9.6 CB buffer.
  • AFP antibody, CEA antibody, CA19-9 antibody, CA242 antibody, CA125 antibody, CA15-3 antibody, CA72-4 antibody, CA50 antibody, SF antibody, POA antibody were diluted with CB buffer to a concentration: 5 ug/ml, 6 ug /ml, 8ug/ml, 8ug/ml, 6ug/ml, 6ug/ml, 5ug/ml, 6ug/ml, 10ug/ml, 10ug/ml.
  • Proclin 300, 0.02% Captisol, and 15% glycerol were diluted to concentrations of 6 ug/ml, 6 ug/ml, 8 ug/ml, 8 ug/ml, 10 ug/ml, 7 ug/ml, 6 ug/ml, 6 ug/ml.
  • CT antibody, SCC antibody, CYFRA21-1 antibody, TSGF antibody were respectively used in 20 mM Tris buffer of pH 8.5 (containing 0.5% PVP, 0.05% Proclin 300, 0.02% Captisol, and 15% glycerol), respectively. It is 6ug/ml, 10ug/ml, 12ug/ml, 10ug/ml.
  • the coated chips were removed, washed 3 times with PBST washing solution of pH 7.4, and then 150 ul of blocking solution (pH 7.4, containing 20% fetal bovine serum, 4% trehalose, 0.01 M PBS, 0.05%) was added to each well. Tween20, 1% PVA and 0.05% sodium azide, the balance of ultrapure water), sealed at room temperature for 1 h, then patted dry, under 15% humidity, room temperature, dried for 4 h, then sealed, stored at 2-8 °C.
  • Biotinylated tumor marker antibody Biotinylation of the above 22 tumor marker antibodies by the aforementioned biotinylation kit;
  • Enzyme dilution containing 0.1 M Tris, 2.5% casein, 2% trehalose, 0.2% sodium p-hydroxybenzoate, 0.01% gum arabic, 1% PEG and 0.05% Proclin 300, the balance being water;
  • Enzyme-labeled streptavidin horseradish peroxidase-labeled streptavidin
  • the horseradish peroxidase-labeled streptavidin and anti-human Ig-HRP are diluted to 6K times with the enzyme standard dilution (6K refers to spicy Root peroxidase-labeled streptavidin concentration and anti-human Ig-HRP concentration).
  • the above-mentioned tumor coated with a tumor marker antibody, a biotinylated tumor marker antibody, an enzyme-labeled streptavidin diluted with an enzyme standard dilution, a color developing solution, and a sample diluent and a washing solution constitute the tumor of the present invention.
  • Marker detection kit for detecting tumor marker antibody, a biotinylated tumor marker antibody, an enzyme-labeled streptavidin diluted with an enzyme standard dilution, a color developing solution, and a sample diluent and a washing solution.
  • the kit of the present invention can detect 100% of the clinical samples of CEA and FPSA at the lower limit of the reference range (compared with the control kit) and compared with the control kit.
  • the correlation between the two can reach R2>0.98 (R>0.99), which indicates that the sensitivity and accuracy of the kit can meet the requirements.
  • 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 was 3% BSA solution, and the enzyme standard dilution was PBS buffer containing 3% BSA, 0.05% Proclin300, pH 7.4 (refer to the existing Patent CN10160396A);
  • 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 5-8.
  • 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 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 6 cells are placed.
  • the monthly detection kit has a detection signal value that is close to 100% compared with the detection signal value of 0 and 3 months, and can maintain the stability at the completion of preparation; compared with the detection signal values of 0, 3, and 6 months. It is still above 90% after being placed for 9 months and can maintain high 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 shows that the 18-month control kit has a significant decrease in the detection signal value.
  • the kit of the present invention and the control kit respectively detect the detection signal values placed at normal temperature for 3, 6, and 9 months, and simultaneously count the ratio of the signal values of the two kits at the same time.
  • the results showed that the control kits that were placed for 6 months showed a significant decrease in the detection signal values of some indicators, while the control kits that were placed for 9 months could not guarantee the stability of the test.
  • the data in Table 6 clearly shows that the stability of the control kit is not as good as the conclusion of the kit of the present invention, and the difference between the two is only in the blocking solution and the enzyme standard dilution.

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Abstract

提供了一种用于酶联免疫试剂盒的组合物以及肿瘤标志物检测试剂盒及其制备方法。所述组合物包括封闭液和酶标稀释液；所述封闭液含有胎牛血清、海藻糖、PBS、Tween20、PVA和叠氮钠；所述酶标稀释液含有Tris、酪蛋白、海藻糖、对羟基苯甲酸钠、阿拉伯树胶、PEG和Proclin300。

Description

一种用于酶联免疫试剂盒的组合物以及肿瘤标志物检测试剂盒及其制备方法
本申请要求于2018年03月07日提交中国专利局、申请号为201810186996.8、发明名称为“一种用于酶联免疫试剂盒的组合物以及肿瘤标志物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肿瘤标志物检测试剂盒及其制备方法。
背景技术
肿瘤，尤其是恶性肿瘤(癌症)是一类严重威胁人类健康的多发病和常见病。近年来，癌症的发病率和死亡率一直呈上升的趋势，而且存在发现晚、治疗不及时，导致死亡率较高的难题。虽随着科学和经济的发展，对肿瘤的治疗发展出了一些新的方法和药物，但及早发现和预防仍然是对癌症的有效防治方法。
肿瘤标志物是指在肿瘤的发生和增殖过程中，由肿瘤细胞本身所产生的或者是由机体对肿瘤细胞反应而产生的，反应肿瘤存在和生长的一类物质，包括蛋白质、激素、酶、多胺及癌基因等。如甲胎蛋白(AFP)、癌胚抗原(CEA)、癌抗原19-9(CA19-9)、癌抗原242(CA242)、癌抗原125(CA125)、癌抗原15-3(CA15-3)、癌抗原72-4(CA72-4)、癌抗原50(CA50)、铁蛋白(SF)、生长激素(HGH)、绒毛膜促性腺激素β亚基(β-HCG)、总前列腺特异性抗原(TPSA)、游离前列腺特异性抗原(FPSA)、降钙素(CT)、鳞状细胞癌抗原(SCC)、神经元烯醇化酶(NSE)、非小细胞肺癌相关抗原(CYFRA21-1)、胃泌素释放肽前体(Pro-GRP)、肿瘤相关物质(TSGF)、胰胚胎抗原(POA)、胃蛋白酶原I(PGI)、胃蛋白酶原II(PGII)。这些肿瘤标志物的检测对肿瘤早期筛查，疗效观察，追踪复发等都具有重要意义。
肿瘤标志物大多无器官特异性，为广谱标志物。例如：正常成人的血液中CEA很难测出，70-90％的结肠腺癌患者CEA高度阳性，在其他恶性肿瘤中的阳性率顺序为胃癌(60-90％)、胰腺癌(70-80％)、小肠腺癌(60-83％)、肺癌(56-80％)、肝癌(62-75％)、乳腺癌(40-68％)、泌尿系癌肿(31-46％)。同时，一种癌症患者往往存在多种肿瘤标志物的异常或是明显升高，因此单项指标检测一般都存在特异性不强、阳性率偏低、特别是对早期肿瘤的检出率不高等难题。同时检测多种肿瘤标志物能显著提高相关肿瘤的检出率，也能更有效的帮助判断肿瘤的类型，以便协助进一步的确诊及诊疗方案的确定。例如：联合测定CEA、PGI、PGII、CA19-9、CA72-4能大大的提高胃癌的检出率；同时检测AFP、SF、CA125有利于肝癌的判断；还有CEA、SCC、ProGRP、CYFRA21-1、NSE、CA125等是肺癌的标志物；等等。鉴于此，对肿瘤标志物多项指标的联合检测逐渐受到多数学者的关注，同时也将受到临床越来越高的重视。多项肿瘤标志物的联合检测尤其适用于健康或亚健康人群的防癌普查，有利于肿瘤的早期发现，为临床诊断、治疗提供检测手段。
目前较常用的肿瘤标志物的检测方法主要有：放射免疫分析法，酶联免疫分析法，化学发光免疫分析法，免疫印迹法等，普遍采用的酶联免疫吸附法虽然灵敏度高、特异性也较好，但检测指标单一，无法达到普查的目的；放免法有污染的危险；化学发光免疫分析法存在发光时间短，需要严格掌握测量时间的缺陷；免疫印迹灵敏度低、操作繁琐，且无法定量测定。随着科技手段的发展和成熟，近来也发展出了部分高通量、高灵敏度的肿瘤标志物抗体芯片试剂盒：CN103869068，其虽然具有廉价、灵敏、准确、高通量、标本使用量少、且能在普通实验室推广和规模化等优点，但该试剂盒需要通过配备标准品、并通过将标准品稀释不同倍数然后测定信号值，来制定标准曲线，用于待测样品的定量，仍然较为繁琐。
我们知道蛋白质芯片是一种高通量的蛋白功能分析技术，是近年来发展起来的一项生物检测技术，这项技术是将蛋白质的分析微缩到小型芯片上，利用荧光或酶显色进行探测，最后用特定的计算机软件加以分 析。如专利CN103869068公开了一种高通量、高灵敏度的肿瘤标志物抗体芯片试剂盒，其虽然具有廉价、灵敏、准确、高通量、标本使用量少、且能在普通实验室推广和规模化等优点，但该试剂盒需要通过配备标准品、并通过将标准品稀释不同倍数然后测定信号值，来制定标准曲线，用于待测样品的定量，仍然较为繁琐。更为值得关注的是，目前该类试剂盒的稳定性并不好。
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，特别是用于检测肿瘤标志物的相关试剂盒；
本发明的另外一个目的在于提供一种包含上述组合物的肿瘤标志物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；所述封闭液含有胎牛血清、海藻糖、PBS、Tween20、PVA和叠氮钠；所述酶标稀释液含有Tris、酪蛋白、海藻糖、对羟基苯甲酸钠、阿拉伯树胶、PEG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(用于稀释酶标记物用)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20％-25％胎牛血清，2％-5％海藻糖，0.01M PBS、0.02％-0.05％Tween20、1％-3％PVA,0.05％叠氮钠，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，20％胎牛血清、4％的海藻糖、0.01M PBS、0.05％Tween20、1％PVA和0.05％叠氮钠，pH值为7.4，余量为超纯水。其中，聚乙烯醇PVA优选为 聚乙烯醇2W。
作为优选，所述酶标稀释液含有0.1M Tris、2.5％-5％酪蛋白、2％-5％海藻糖、0.2％-0.5％对羟基苯甲酸钠、0.01％-0.05％阿拉伯树胶、1％-5％PEG，0.05％proclin300，余量为水。所述百分比中除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 Tris、2.5％酪蛋白、2％海藻糖、0.2％对羟基苯甲酸钠、0.01％阿拉伯树胶、1％PEG和0.05％Proclin300，余量为水。其中，聚乙二醇优选为聚乙二醇1W。
本发明利用上述组合物进行肿瘤标志物抗体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肿瘤标志物抗体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较高稳定性，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6个月后仍然能够保持制备完成时的稳定性，在放置9个月后仍然能够保持较高的稳定性，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，在放置9个月后已经无法保证检测的稳定性了。
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，特别是在制备肿瘤标志物酶联免疫检测试剂盒中的应用。
同时，本发明还提供一种肿瘤标志物检测试剂盒，包括以下组分：
包被有肿瘤标志物抗体的蛋白芯片、生物素化的肿瘤标志物抗体、用酶标稀释液稀释包含酶标亲和素的酶标记物、样品稀释液、洗涤液和显色液；其中，所述蛋白芯片包被肿瘤标志物后采用封闭液封闭，所述封闭液含有胎牛血清、海藻糖、PBS、Tween20、PVA和叠氮钠，所述酶标稀释液含有Tris、酪蛋白、海藻糖、对羟基苯甲酸钠、阿拉伯树胶、PEG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，酶标记物中，酶标亲和素为辣根过氧化物酶标记的亲和素。其中，亲和素优选为链霉亲和素。作为进一步优选，所述酶标物中还包括抗人Ig-HRP，可用于有样品质控点的蛋白芯片使用。
作为优选，所述肿瘤标志物选自AFP、CEA、CA19-9、CA242、CA125、CA15-3、CA72-4、CA50、SF、HGH、β-HCG、TPSA、FPSA、CT、SCC、NSE、CYFRA21-1、Pro-GRP、TSGF、POA、PGI、PGII中的一种或两种以上。在本发明具体实施方式中，本发明选择上述22种肿瘤标志物。
在包被中，所述肿瘤标志物采用CB缓冲液、PBS缓冲液或Tris缓冲液为肿瘤标志物稀释缓冲液进行包被；作为优选，所述缓冲液选自pH9.6的CB缓冲液、pH7.4的PBS缓冲液(优选0.01M)或pH8.5的Tris缓冲液(优选20mM)，更优选地，在缓冲液中添加PEG或PVP，甘油和Proclin300，同时添加了水溶性的环糊精，可使包被更稳定、肿瘤标志物包被点更规则、更圆，CV更小。
其中，水溶性环糊精可以是Captisol、2-羟基-β-环糊精或羧甲基-β-环糊精等，浓度为0.02％；PEG的浓度为5％，PVP的浓度为0.5-0.6％，甘油的浓度为15％，Proclin300浓度为0.05％，所述百分比中除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，CT抗体、SCC抗体、CYFRA21-1抗体、TSGF抗体的稀释缓冲液为PH8.5的20mM Tris缓冲液(含0.5％的PVP、0.05％的Proclin300，0.02％Captisol，以及15％的甘油)，终浓度分别为6ug/ml、10ug/ml、12ug/ml、10ug/ml。
AFP抗体、CEA抗体、CA19-9抗体、CA242抗体、CA125抗体、CA15-3抗体、CA72-4抗体、CA50抗体、SF抗体、POA抗体的稀释缓冲液为pH9.6的CB缓冲液，终浓度分别为5ug/ml、6ug/ml、8ug/ml、8ug/ml、6ug/ml、6ug/ml、5ug/ml、6ug/ml、10ug/ml、10ug/ml。
HGH抗体、β-HCG抗体、TPSA抗体、FPSA抗体、NSE抗体、Pro-GRP抗体、PGI抗体、PGII抗体的稀释缓冲液为PH7.4的0.01M的PBS缓冲液(含有0.6％的PVP、0.05％的Proclin300、0.02％Captisol，以及15％的甘油)，终浓度分别为6ug/ml、6ug/ml、8ug/ml、8ug/ml、10ug/ml、7ug/ml、6ug/ml、6ug/ml。
上述各肿瘤标志物抗体优选为单克隆抗体。
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、 参考临界值点、酶标质控点、标准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个标准曲线点(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，阳性质控点可以是一定浓度的生物素化的BSA，而阴性质控点可以是低于反映信号值的微量浓度的生物素化的BSA，或采用其他的无关蛋白来替代；酶标质控点可以是生物素化的BSA，或生物素化的其他无关蛋白；样品点质控点(SC)和标准曲线点分别是不同浓度的生物素化的BSA。芯片本身的位置参考点(Loc)是生物素化的BSA，主要是对阵列的定位作用。
本发明所述酶标记亲和素中的酶可选择常规的酶以及对应的显色液，如辣根过氧化物酶。
本发明使用的显色液是增强型化学发光底物(ECL)，通过荧光检测装置或仪器进行反应结果的读取。在其他实施例中也可以使用其他的显色底物，如p-NPP，TMB等。
本发明还对应提供了所述试剂盒的制备方法，包括：
将肿瘤标志物抗体在蛋白芯片上进行包被，包被后洗涤，然后加入封闭液封闭，获得包被有肿瘤标志物抗体的蛋白芯片，所述封闭液含有胎牛血清、海藻糖、PBS、Tween20、PVA和叠氮钠；
配制含有Tris、酪蛋白、海藻糖、对羟基苯甲酸钠、阿拉伯树胶、PEG和Proclin300的酶标稀释液并稀释包含酶标亲和素的酶标记物，获得用酶标稀释液稀释的酶标记亲和素，然后制备生物素化的肿瘤标志物抗体以及配制样品稀释液、洗涤液和显色液，获得肿瘤标志物检测试剂盒。
采用本发明肿瘤标志物检测试剂盒检测血清样本，对CEA和FPSA的处于参考范围下限(cutoff值)的临床样本的检出率都能达到100％(与 现有市售试剂盒比较)，且与市售试剂盒比较两者的相关性均能达到R2>0.98(R>0.99)，可以说明本发明试剂盒的准确度和灵敏度均达到预期要求。
此外，不管是CEA还是AFP在批内还是批间的检测结果，其CV值都低于10％，表明本发明试剂盒精密度高。
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肿瘤标志物检测试剂盒在具有较佳稳定性的基础上，还具备较高的灵敏度、准确度和精密度。
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肿瘤标志物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肿瘤标志物检测试剂盒及其制备方法做进一步说明。
实施例1：本发明所述肿瘤标志物检测试剂盒的制备
1、抗肿瘤标志物抗体和相关蛋白的包被
蛋白芯片阵列中的Loc、EC、PC、NC、S1、S2、S3、分别包被的是2ug/ml、2ug/ml、2ug/ml、0.01ug/ml、0.5ug/ml、2ug/ml、4ug/ml的生物素化(Thermo的
Figure PCTCN2018093059-appb-000001
Sulfo-NHS-LC-Biotin生物素化试剂盒)的BSA，可用PH9.6的CB缓冲液(含5％的PEG400、0.05％的Proclin300，以及0.02％的Captisol)进行稀释。
样品质控点使用是1.5ug/ml抗人IgG，稀释缓冲液为PH9.6的CB缓冲液。
AFP抗体、CEA抗体、CA19-9抗体、CA242抗体、CA125抗体、CA15-3抗体、CA72-4抗体、CA50抗体、SF抗体、POA抗体，分别用CB缓冲液稀释至浓度：5ug/ml、6ug/ml、8ug/ml、8ug/ml、6ug/ml、6ug/ml、5ug/ml、6ug/ml、10ug/ml、10ug/ml。
HGH抗体、β-HCG抗体、TPSA抗体、FPSA抗体、NSE抗体、Pro-GRP抗体、PGI抗体、PGII抗体，分别用PH7.4的0.01M的PBS缓冲液(含有0.6％的PVP、0.05％的Proclin300、0.02％Captisol，以及15％的甘油)稀释至浓度：6ug/ml、6ug/ml、8ug/ml、8ug/ml、10ug/ml、7ug/ml、6ug/ml、6ug/ml。
CT抗体、SCC抗体、CYFRA21-1抗体、TSGF抗体，分别用PH8.5的20mM Tris缓冲液(含0.5％的PVP、0.05％的Proclin300，0.02％Captisol，以及15％的甘油)，终浓度分别为6ug/ml、10ug/ml、12ug/ml、10ug/ml。
所有稀释好的蛋白分别用0.22um的滤膜过滤，然后利用BioDot精密点样仪按要求进行阵列的包被，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
Figure PCTCN2018093059-appb-000002
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(pH7.4，含20％胎牛血清、4％的海藻糖、0.01M PBS、0.05％Tween20、1％PVA和0.05％叠氮钠，余量超纯水)，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。
3、配制生物素化的肿瘤标志物抗体、酶标稀释液、酶标记链霉亲和素、显色液、样品稀释液和浓缩洗涤液
生物素化的肿瘤标志物抗体：用前述生物素化试剂盒分别对以上22种肿瘤标志物抗体进行生物素化；
酶标稀释液：含有0.1M Tris、2.5％酪蛋白、2％海藻糖、0.2％对羟基苯甲酸钠、0.01％阿拉伯树胶、1％PEG和0.05％Proclin300，余量为水；
酶标记链霉亲和素：辣根过氧化物酶标记的链霉亲和素；
使用时，用酶标稀释液将辣根过氧化物酶标记的链霉亲和素与抗人Ig-HRP(辣根过氧化物酶标记的抗人IgG)稀释至6K倍混合(6K指辣根过氧化物酶标记的链霉亲和素浓度以及抗人Ig-HRP浓度)。
显色液：ECL。
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。
上述包被有肿瘤标志物抗体的蛋白芯片、生物素化的肿瘤标志物抗体、酶标稀释液稀释的酶标记链霉亲和素、显色液、以及样品稀释液和洗涤液组成本发明肿瘤标志物检测试剂盒。
4、检测方法
(1)取出芯片和相关试剂，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)加检测抗体：每孔加入100uL生物素化的肿瘤标志物抗体。
(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)加酶标记物(酶标记链酶亲和素和酶标抗人IgG混合液)：每孔加入100uL酶标记物。
(7)温育：室温静置反应30min。加300uL洗涤液，洗涤3次，每次静置1min。
(8)显色：每孔加入ECL显色剂50uL，室温静置，避光反应30min。
(9)测定：30min内，用化学发光芯片分析仪读取并分析每个反应孔对应检测指标的信号值，通过NC、CO、以及S1-S3五点校准的标准曲线，来计算并判断结果的阴阳性及强度。
实施例2：临床样本的检测
1、灵敏度和准确度
以检测CEA和FPSA为例，选取7个临床样本，以湖州数康生物科技有限公司的蛋白芯片试剂盒作为对照，与实施例1试剂盒同时检测临床样本，具体的实验结果和数据如下表：
表2
Figure PCTCN2018093059-appb-000003
由以上结果分析可知，本发明的试剂盒对CEA和FPSA的处于参考范围下限(cutoff值)的临床样本的检出率都能达到100％(与对照试剂盒比较)，且与对照试剂盒比较两者的相关性均能达到R2>0.98(R>0.99)，可以说明试剂盒的灵敏性和准确度均能达到要求。
2、精密度
以检测CEA和AFP为例，选取2个临床样本，进行批内精密度检测；同时选取3个样本，进行批间精密度检测，结果见表3和表4。
表3
Figure PCTCN2018093059-appb-000004
Figure PCTCN2018093059-appb-000005
表4
Figure PCTCN2018093059-appb-000006
Figure PCTCN2018093059-appb-000007
由以上精密度测试结果可以看出，不管是CEA还是AFP在批内还是批间的检测结果，其CV值都低于10％，精密度高。
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，区别在于封闭液采用3％的BSA溶液，酶标稀释液采用含3％的BSA、0.05％Proclin300的PBS缓冲液，PH7.4(参考现有专利CN10160396A)；
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5-8。
1、实施例1试剂盒低温下的稳定性数据
表5
Figure PCTCN2018093059-appb-000008
Figure PCTCN2018093059-appb-000009
Figure PCTCN2018093059-appb-000010
由表5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6
Figure PCTCN2018093059-appb-000011
Figure PCTCN2018093059-appb-000012
Figure PCTCN2018093059-appb-000013
由表6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6个月的试剂盒，其检测信号值与放置0、3个月的检测信号值相比，仍接近100％能够保持制备完成时的稳定性；与0、3、6个月的检测信号值相比，在放置9个月后仍然处于90％以上，能够保持较高的稳定性。
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7
Figure PCTCN2018093059-appb-000014
Figure PCTCN2018093059-appb-000015
由表7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5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8
Figure PCTCN2018093059-appb-000016
Figure PCTCN2018093059-appb-000017
由表8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，其某些指标的检测信号值开始出现显著的下降，而放置了9个月的对照试剂盒，其已经无法保证检测的稳定性了，结合表6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；所述封闭液含有胎牛血清、海藻糖、PBS、Tween20、PVA和叠氮钠；所述酶标稀释液含有Tris、酪蛋白、海藻糖、对羟基苯甲酸钠、阿拉伯树胶、PEG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20％-25％胎牛血清，2％-5％海藻糖，0.01M PBS、0.02％-0.05％Tween20、1％-3％PVA,0.05％叠氮钠，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 Tris、2.5％-5％酪蛋白、2％-5％海藻糖、0.2％-0.5％对羟基苯甲酸钠、0.01％-0.05％阿拉伯树胶、1％-5％PEG，0.05％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肿瘤标志物免疫检测试剂盒。
  6. 一种肿瘤标志物检测试剂盒，其特征在于，包括以下组分：
    包被有肿瘤标志物抗体的蛋白芯片、生物素化的肿瘤标志物抗体、用酶标稀释液稀释包含酶标亲和素的酶标记物、样品稀释液、洗涤液和显色液；其中，所述蛋白芯片包被肿瘤标志物后采用封闭液封闭，所述封闭液含有胎牛血清、海藻糖、PBS、Tween20、PVA和叠氮钠，所述酶标稀释液含有Tris、酪蛋白、海藻糖、对羟基苯甲酸钠、阿拉伯树胶、PEG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20％-25％胎牛血清，2％-5％海藻糖，0.01M PBS、0.02％-0.05％Tween20、1％-3％PVA,0.05％叠氮钠，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 Tris、2.5％-5％酪蛋白、2％-5％海藻糖、0.2％-0.5％对羟基苯甲酸钠、0.01％-0.05％阿拉伯树胶、1％-5％PEG，0.05％proclin300，余量为水。
  9. 根据权利要求6所述试剂盒，其特征在于，所述肿瘤标志物选自AFP、CEA、CA19-9、CA242、CA125、CA15-3、CA72-4、CA50、SF、HGH、β-HCG、TPSA、FPSA、CT、SCC、NSE、CYFRA21-1、Pro-GRP、TSGF、POA、PGI、PGII中的一种或两种以上。
  10. 根据权利要求6或9所述试剂盒，其特征在于，所述肿瘤标志物采用CB缓冲液、PBS缓冲液或Tris缓冲液为肿瘤标志物稀释缓冲液进行包被。
  11. 根据权利要求6-10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、标准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：
    将肿瘤标志物抗体在蛋白芯片上进行包被，包被后洗涤，然后加入封闭液封闭，获得包被有肿瘤标志物抗体的蛋白芯片，所述封闭液含有胎牛血清、海藻糖、PBS、Tween20、PVA和叠氮钠；
    配制含有Tris、酪蛋白、海藻糖、对羟基苯甲酸钠、阿拉伯树胶、PEG和Proclin300的酶标稀释液并稀释包含酶标亲和素的酶标记物，获得用酶标稀释液稀释的酶标记亲和素，然后制备生物素化的肿瘤标志物抗体以及配制样品稀释液、洗涤液和显色液，获得肿瘤标志物检测试剂盒。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023035825A1 (zh) * 2021-09-13 2023-03-16 苏州长光华医生物医学工程有限公司 抗ca724抗体或其抗原结合片段及其制备方法和应用

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109212191A (zh) * 2018-09-27 2019-01-15 郑州安图生物工程股份有限公司 一种用于检测细胞角蛋白19片段的试剂盒
CN109270283A (zh) * 2018-10-18 2019-01-25 郑州标源生物科技有限公司 一种性能稳定的肿瘤标志物复合质控品
CN110954693A (zh) * 2019-11-29 2020-04-03 郑州大学 一种肿瘤标志物Cyfra21-1的Simoa试剂盒及其应用
CN113238038B (zh) * 2021-05-11 2021-12-17 图凌(杭州)生物医药有限公司 用于免疫组织化学检测的抗体稀释液及其制备方法和应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101308137A (zh) * 2007-05-15 2008-11-19 广东省疾病预防控制中心 登革病毒IgG抗体酶联免疫诊断试剂盒
CN101308138A (zh) * 2007-05-15 2008-11-19 广东省疾病预防控制中心 登革病毒IgM抗体酶联免疫诊断试剂盒
CN102368070A (zh) * 2011-07-01 2012-03-07 上海永昶医学诊断用品有限公司 检测人血浆中mr-1s含量的试剂盒及其制备方法
EP3052936A1 (en) * 2013-09-23 2016-08-10 Assaypro, LLC Immunoassays using over-labeled fluorescent probes

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1235047C (zh) * 2002-10-24 2006-01-04 上海数康生物科技有限公司 一种多指标并行检测的蛋白芯片检测系统及制备方法
CN1913785A (zh) * 2004-01-30 2007-02-14 巴斯福股份公司 稳定酶制剂
US8236519B2 (en) * 2008-04-28 2012-08-07 Sekisui Diagnostics, Llc Acetaminophen assay kit
CN101622541A (zh) * 2008-10-24 2010-01-06 韩国帕克特生物科技有限公司 糖化血红蛋白定量检测系统及使用该系统检测糖化血红蛋白含量的方法
US20150299760A1 (en) * 2012-11-21 2015-10-22 Oslo Universitetssykehus Hf Systems and methods for monitoring biological fluids
WO2015017285A2 (en) * 2013-07-30 2015-02-05 Bio-Rad Laboratories, Inc. Multiplex blocker beads for immunoassays
EP3306318A4 (en) * 2015-05-29 2019-03-06 Kyocera Corporation DETECTION METHOD AND DETECTION DEVICE
CN106814190A (zh) * 2017-01-19 2017-06-09 深圳市雷诺华科技实业有限公司 Pct与crp联检试纸条及其制备方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101308137A (zh) * 2007-05-15 2008-11-19 广东省疾病预防控制中心 登革病毒IgG抗体酶联免疫诊断试剂盒
CN101308138A (zh) * 2007-05-15 2008-11-19 广东省疾病预防控制中心 登革病毒IgM抗体酶联免疫诊断试剂盒
CN102368070A (zh) * 2011-07-01 2012-03-07 上海永昶医学诊断用品有限公司 检测人血浆中mr-1s含量的试剂盒及其制备方法
EP3052936A1 (en) * 2013-09-23 2016-08-10 Assaypro, LLC Immunoassays using over-labeled fluorescent probes

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023035825A1 (zh) * 2021-09-13 2023-03-16 苏州长光华医生物医学工程有限公司 抗ca724抗体或其抗原结合片段及其制备方法和应用

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