WO2016019797A1 - 检测血清糖蛋白岩藻糖指数的化学发光蛋白芯片、试剂盒及检测方法 - Google Patents

检测血清糖蛋白岩藻糖指数的化学发光蛋白芯片、试剂盒及检测方法 Download PDF

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WO2016019797A1
WO2016019797A1 PCT/CN2015/084356 CN2015084356W WO2016019797A1 WO 2016019797 A1 WO2016019797 A1 WO 2016019797A1 CN 2015084356 W CN2015084356 W CN 2015084356W WO 2016019797 A1 WO2016019797 A1 WO 2016019797A1
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afp
detection
serum
protein
alpha
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PCT/CN2015/084356
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English (en)
French (fr)
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李宁
张爱英
王升启
柯杨
张永宏
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首都医科大学附属北京佑安医院
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Priority to SE1750227A priority Critical patent/SE1750227A1/sv
Priority to KR1020177006041A priority patent/KR20170040318A/ko
Priority to KR1020187009587A priority patent/KR101914673B1/ko
Priority to GB1703351.5A priority patent/GB2548978B/en
Publication of WO2016019797A1 publication Critical patent/WO2016019797A1/zh
Priority to AU2016101432A priority patent/AU2016101432A4/en

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    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
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Definitions

  • the invention relates to protein detection technology, in particular to a chemiluminescent protein chip and a detection method for detecting serum glycoprotein fucose index.
  • Alpha fetoprotein (AFP) produced by benign liver disease such as primary liver cancer and hepatitis and cirrhosis has a large difference in its sugar chain structure. Compared with benign liver disease, AFP produced by liver cancer has fucoids. The sugar index is much higher. Fucose has the property of binding to lentil. AFP can be classified into AFP-L1, AFP-L2 and AFP-L3 according to its affinity for gluten agglutinin. Among them, AFP-L1 is mainly from benign liver disease, AFP-L2 is mainly from pregnant women, and AFP-L3 is a fucose glycosylation form of alpha-fetoprotein, mainly derived from HCC. In 2005, the FDA officially approved AFP-L3 as one of the markers of primary liver cancer. AFP-L3 has high specificity and sensitivity in early diagnosis, differential diagnosis, efficacy evaluation and prognosis monitoring of liver cancer.
  • Fucose is a methylated hexose, which is found in various glycoprotein sugar chains in tissues and serum, and is called protein-bound fucose (P-bf). There is a fucose residue on the AFP carbohydrate chain. This heterogene is called fucosylated AFP (FucAFP), and its percentage of total AFP is called fucosylation index (Fuol). .
  • the fucosylation index has important theoretical significance and clinical application significance, and can be used as an important index in the diagnosis and prognosis of liver cancer.
  • the present invention provides a kit suitable for quantitative detection of alpha-fetoprotein and/or fucosylated alpha-fetoprotein in biological samples based on the needs and gaps in the field of quantitative detection techniques for AFP and AFP-L3 in serum. And detection method, the program It is not only suitable for detecting AFP antigen in serum, but also has the advantages of being versatile for detecting other fucosylated proteins, and has the advantages of time saving, economy, accuracy and convenience.
  • a chemiluminescent protein chip for detecting a serum glycoprotein fucose index characterized in that: a matrix slide of the protein chip comprises at least one detection sub-region, and one detection sub-region detects a serum sample;
  • detection plaque areas There are two detection plaque areas and one platoon control plaque area in the detection sub-region, one of which detects the detection spot formed by the specific antibody of immobilized alpha-fetoprotein, and the other detection area has the fixed lentil formation.
  • a test spot having a control spot formed by immobilizing bovine serum albumin;
  • the concentration of the substance on all the detection spots in the same detection spot area is the same.
  • the one detection spot area includes at least two of the detection spots.
  • the specific antibody to alpha-fetoprotein is a murine anti-human alpha-fetoprotein antibody.
  • the substrate slide has a plurality of the detection sub-regions, each of the detection plaque regions comprising four detection plaques arranged in a row, the control plaque region comprising four control plaques arranged in a row;
  • the detection spots and the control spots are arranged in three parallel rows.
  • a protrusion is provided between the detection sub-regions as a physical partition.
  • a chemiluminescence kit for detecting a serum glycoprotein fucose index comprising the chemiluminescent protein chip according to any one of claims 1 to 4.
  • AFP standards biotinylated AFP polyclonal antibodies, avidin HRP and HRP chemiluminescent substrate solutions; the biotinylated AFP polyclonal antibody is a rabbit derived antibody, and is immobilized on the detection spot AFP-specific antibodies are derived from different species.
  • kits for detecting alpha-fetoprotein and/or fucosylated alpha-fetoprotein and/or serum glycoprotein fucose index are used for detecting alpha-fetoprotein and/or fucosylated alpha-fetoprotein and/or serum glycoprotein fucose index.
  • a method for quantitatively detecting fucosylated protein characterized in that any of the above chemiluminescent protein chips is used, comprising the following steps:
  • the serum sample to be tested is diluted and added to the detection sub-region of the chemiluminescent protein chip, and after incubation, the detection sub-region is washed with PBST to remove the non-specific conjugate;
  • Biotin-labeled AFP antibody diluted in PBS was added, and after incubation, washed with PBST to remove non-specific conjugates;
  • the abscissa x of the alpha-fetoprotein standard curve equation is the gradient concentration value of the AFP standard;
  • the ordinate y is the AFP standard of the shaving concentration as a series of samples to be tested, and the method detected by the method of the step (1) a series of luminescent pixel values of fetal protein;
  • the abscissa x of the fucosylated protein standard curve equation is the gradient concentration value of AFP-L3 in the AFP-L3 standard; the ordinate y is a series of samples to be tested with the AFP-L3 standard of the shaving concentration. a series of luminescent pixel values of the obtained fucosylated protein detected by the method of the step (1); the AFP-L3 standard is a serum containing fucosyl protein (AFP);
  • the luminescence pixel value of alpha fetoprotein in the serum sample to be tested in step (1) is taken into the alpha-fetoprotein standard curve equation to calculate the alpha-fetoprotein concentration of the diluted serum, multiplied by the dilution factor to obtain a test Serum alpha-fetoprotein concentration;
  • the luminescence pixel value of the fucosylated protein of the serum sample to be tested in step (1) is taken into the fucosylated protein standard curve equation to calculate the degraded serum of the fucoid
  • concentration of the glycosylated protein is multiplied by the dilution factor to obtain the concentration of the serum fucosylated protein to be tested;
  • the ratio fucosylation index of the ratio of the serum fucosylated protein to be tested to the alpha-fetoprotein concentration of the test serum is the ratio fucosylation index of the ratio of the serum fucosylated protein to be tested to the alpha-fetoprotein concentration of the test serum.
  • the incubation refers to incubation for 30 minutes at 37 °C.
  • the invention provides a chemiluminescent protein chip for detecting serum glycoprotein fucose index, based on the principle of antibody antigen-antibody sandwich reaction and chemiluminescence principle, and immobilized with alpha-fetoprotein-specific antibody and lentil, specific for alpha-fetoprotein
  • the antibody was used to bind all alpha-fetoproteins (AFP-L1, AFP-L2 and AFP-L3) in serum, and lentil was used to bind fucosylated alpha-fetoprotein.
  • a control spot is also set at the same time.
  • the total concentration of alpha-fetoprotein and the concentration of fucosylated alpha-fetoprotein in the serum to be tested can be simultaneously detected under absolutely the same conditions, and the serum glycoprotein fucose index can be accurately obtained.
  • the chemiluminescent protein chip provided by the invention comprises at least one sub-detection zone for detecting a blood sample. In most embodiments, at least two detection sub-regions are preferably provided, one for detecting the control serum and the other for detecting the blood sample to be tested.
  • one of the detection sub-regions includes four detection spots to which AFP-specific antibodies are immobilized, four detection spots to which lentils are immobilized, and four control spots.
  • the two detection spots and the control spots are arranged in three parallel rows.
  • the present invention also provides a chemiluminescent kit for detecting a serum glycoprotein fucose index, which comprises the above protein chip and a conventional reagent for chemiluminescence, standard curve equation data and the like.
  • the use of the protein chip of the present invention requires a large reduction in the amount of blood and antibodies, only the original serum volume of 2.5ul ⁇ 10ul, while the ELISA method requires serum 50ul; protein chip plate antibody spotting, 5ul at least can be sampled 20
  • the chip which detects 200 sera, requires far less antibody than the ELISA method, greatly reducing the cost and cost of testing.
  • the present invention also provides a method for quantitatively detecting fucosylated alpha-fetoprotein using the kit.
  • the present invention adopts the purchased AFP antigen standard to prepare a gradient concentration of the AFP diluted solution to be tested, and determines the illuminating pixel value corresponding to each gradient by the chemiluminescence detection method, with the gradient concentration as the abscissa and the fluorescence pixel value as the ordinate.
  • the method for detecting the serum glycoprotein fucose index provided by the invention is characterized in that the antibody and the antigen are specifically combined and the lentilin is specifically combined on the above protein chip, and the serum or plasma sample is added for incubation, and then the organism is added.
  • the labeled AFP polyclonal antibody, HRP-labeled avidin, and finally HRP luminescent substrate were added, and the luminescent signal was scanned and quantified by a chemiluminescence scanner. The obtained signal value was taken into a pre-made linear regression equation to obtain the concentration of fucose protein AFP-L3 in the sample.
  • the detection principle of the method of the present invention is different from the general chemiluminescence immunoreaction.
  • the usual chemiluminescence immunoreactive Elisa reaction forms an "antibody-antigen-horseradish peroxidase-labeled secondary antibody" complex, and finally HRP chemiluminescence is added.
  • the substrate solution obtained a luminescence value.
  • the secondary antibody in the present invention is labeled with horseradish peroxidase, the sugar residue of horseradish peroxidase binds to lentil, thereby Serious interference with the detection value, some experiments done by the present invention prove that the accurate fucosylation index is not obtained, the false positive is very high, and the normal serum can detect a very high fucosylation index.
  • the principle of the chip and method provided by the present invention is as follows: the anti-AFP monoclonal antibody and lentin are sequentially immobilized on a protein chip, and the serum to be tested, the biotin-labeled AFP polyclonal antibody and the avidin are sequentially added.
  • HRP forms "AFP monoclonal antibody-AFP-biotin-labeled AFP polyclonal antibody-Avidin HRP complex" and "Larmatin-AFP-L3-Biotin-labeled AFP antibody-avidin HRP complex", respectively.
  • HRP chemiluminescence substrate solution was added for incubation, and the luminescence pixel value was obtained by scanning with a chemiluminometer. The pixel value was substituted into the linear regression equation corresponding to the standard curve to obtain the concentration of AFP and AFP-L3, respectively, thereby obtaining the fucoid.
  • the experimental results prove that the method of the present invention can not only perform qualitative detection, but also quantitatively detect AFP and fucosylated AFP by luminous intensity. Compared with the ELISA method, the sensitivity and specificity are superior to the ELISA method. From the time comparison, the ELISA test requires at least 3 hours, and the present invention requires only 1.5 hours; from the comparison of the amount of the antibody, the antibody is spotted by using the protein chip in the kit of the invention, and 5 ul of the antibody can be at least 20 chips. 200 sera were tested, and the antibody requirement was much lower than the ELISA method; compared with the serum dosage, the ELISA method required 50 ul of serum, and the kit and the detection method of the present invention only required 2.5 ul to 10 ul of the original sample. The amount of serum; therefore, the kit and the detection method provided by the invention have the characteristics of high sensitivity, time saving, economy, etc., and can greatly reduce the cost and time of blood protein detection.
  • the method of the present invention combines the application of chemiluminescence detection method, standard curve and protein chip technology to ensure high sensitivity, accuracy, high efficiency and low cost of AFP-L3 quantitative detection using the kit.
  • the detection method provided by the invention is a feasible, reliable, economical, simple and time-saving method.
  • the technical scheme of the present invention will provide an economical and reliable kit and detection method for large-scale, high-throughput detection of fucosylated alpha-fetoprotein in serum.
  • FIG. 1 Schematic diagram of AFP/lentin protein chip spotting
  • Figure 2 Flow chart of AFP/lentin antibody sandwich protein chip
  • the spotted antibodies of the protein chip were different concentrations of AFP antibody, A: 1 mg/ml; B: 0.5 mg/ml; C: 0.25 mg/ml; the test substances were 1.80 ng/ml, 2.40 ng/ml, 3.20 ng/ Ml, 4.10ng/ml, 5.5ng/ml, 6. liver cancer serum, 7. liver cancer serum, 8. blank control, 9. healthy serum, 10. liver cancer serum.
  • test substances were (1-5) 80 ng/ml, 40 ng/ml, 20 ng/ml, 10 ng/ml, 5 ng/ml, 6. liver cancer serum, 7. liver cancer serum, 8 liver cancer serum, 9 liver cancer serum, 10 liver cancer.
  • Serum chip antibody spotting antibody is AFP 0.5mg/ml
  • A AFP antibody 0.5 mg/ml
  • B lentil 4 mg/ml
  • spotted different concentrations of serum concentration of AFP-L3 (1-5) 100 ng/ml; 50 ng/ml; 25 ng/ml; 12.5 ng/ Ml; 6.25 ng/ml; (6-9) 100 ng/ml; 50 ng/ml; 25 ng/ml; 12.5 ng/ml. (10) Blank control.
  • Figure 8 AFP/lentin sampling chip for scanning liver cancer and normal serum samples.
  • Chemiluminescence scanner developed by the Academy of Military Medical Sciences.
  • Mouse monoclonal antibody AFP (Shenzhen Fei Peng Company), lentin (Sigma), aldehyde-based chip (Shanghai Bain Company), biotin-labeled rabbit-derived antibody, HRP-labeled avidin (American abcam) , HRP chemiluminescence substrate liquid A and liquid B, mixed according to 1:1 ratio, freshly arranged. (Millipore, USA).
  • PBS formula: sodium chloride (NaCl) 8g, potassium chloride (KCl) 0.2g, disodium hydrogen phosphate (Na2HPO4) 1.44g, potassium dihydrogen phosphate (KH2PO4) 0.24g, adjusted to pH 7.4, constant volume 1L
  • the chip is an aldehyde-based chip (Shanghai Bain Company), each chip contains 10 test squares (detection sub-area), one serum is tested for each square, and 10 serums are tested at a time.
  • mice monoclonal antibody AFP (Shenzhen Fei Peng Company) and lentin Sigma) were sequentially placed on the chip, and the concentration was monoclonal antibody AFP 0.5 mg/ml.
  • Lentin 4mg/ml pointted into two rows of eight test spots; 10% bovine serum albumin (BSA) as a negative control, also sampled four times, point into a control spot
  • the prepared protein chip was used to detect tumor markers in dynamic serum samples of healthy control group and liver cancer experimental group.
  • the cells were washed 4 times with PBST to remove non-specific binding, and then PBS-diluted biotin-labeled rabbit primary antibody was added and incubated at 37 ° C for 30 minutes.
  • the rabbit-derived antibody binds to the antigen to form a murine antibody-AFP-rabbit-derived biotin-labeled antibody complex, and a lentil-fucosylated AFP-rabbit-derived biotin-labeled antibody complex.
  • the cells were washed 4 times with PBST to remove non-specific binding, and then added with PBS diluted avidin HRP, and incubated at 37 ° C for 30 minutes. Biotin binds to avidin to form "murine antibody-AFP-rabbit biotinylated antibody-avidin HRP complex", and "smectin-fucosylated AFP-rabbit biotinylated antibody - avidin HRP complex".
  • the cells were washed 4 times with PBST to remove non-specific binding, HRP luminescent substrate was added, incubated at 37 ° C for 30 minutes, and scanned by a chemiluminescence scanner.
  • the chemiluminescent pixel on the solid phase carrier is positively correlated with the amount of the antigen to be detected in the specimen. At this time, the pixel value in the complex is determined to determine the antigen content to be tested.
  • the chip spotting antibody (mouse primary antibody) and the detection antibody (rabbit primary antibody) were taken from animals of different species.
  • Figure 2 shows the flow chart of the antibody sandwich protein chip.
  • AFP antigen American abcam company
  • concentration gradients (1-5) 80 ng/ml, 40 ng/ml, 20 ng/ml, 10 ng/ml, 5 ng/ml, 6.
  • the concentration gradients of the AFP standards were measured using the procedure and the protein chip in Example 1.
  • the detection results are shown in Fig. 3.
  • the test results are plotted as a standard curve, with the concentration of the standard as the abscissa and the pixel value as the ordinate, and the standard curve is drawn on the coordinate paper. Find the corresponding concentration from the standard curve according to the pixel value of the sample; multiply by the dilution factor; or calculate the linear regression equation of the standard curve by using the concentration of the standard and the OD value, and substitute the OD value of the sample into the equation to calculate the sample concentration. Then multiply by the dilution factor, which is the actual concentration of the sample.
  • the standard curve and the regression equation a are shown in Fig. 4.
  • AFP antigen American abcam company
  • concentration gradients (1-5) 80 ng/ml, 40 ng/ml, 20 ng/ml, 10 ng/ml, 5 ng/ml, 6.
  • the concentration gradients of the AFP standards were measured using the procedure and the protein chip in Example 1.
  • the detection results are shown in Fig. 3.
  • the test results are plotted as a standard curve, with the concentration of the standard as the abscissa and the pixel value as the ordinate, and the standard curve is drawn on the coordinate paper. Find the corresponding concentration from the standard curve according to the pixel value of the sample; multiply by the dilution factor; or use the standard
  • the linear regression equation of the standard curve is calculated from the concentration and OD value of the quasi-object. The OD value of the sample is substituted into the equation to calculate the sample concentration, and then multiplied by the dilution factor, which is the actual concentration of the sample.
  • the standard curve and the regression equation a are shown in the figure. 4.
  • Serum with known AFP-L3 concentration diluted by dilution, set to different concentration gradients, (1-5) 200 ng / ml, 100 ng / ml, 50 ng / ml, 25 ng / ml, 12.5 ng / ml, (6- 9) 200 ng/ml, 100 ng/ml, 50 ng/ml, 25 ng/ml. 10 blank control ( Figure 6).
  • the concentration gradient of the serum (AFP-L3) standard was detected by the procedure of Example 1 and the protein chip, and the detection results are shown in Fig. 6.
  • the test results are plotted as a standard curve, with the concentration of the standard as the abscissa and the pixel value as the ordinate, and the standard curve is drawn on the coordinate paper. Find the corresponding concentration from the standard curve according to the pixel value of the sample; multiply by the dilution factor; or calculate the linear regression equation of the standard curve by using the concentration of the standard and the OD value, and substitute the OD value of the sample into the equation to calculate the sample concentration. Then multiply by the dilution factor, which is the actual concentration of the sample.
  • the standard curve and the regression equation b are shown in Fig. 7.
  • liver cancer sera from the specimen library of You'an Hospital affiliated to Capital Medical University;
  • the detection process is the same as in the embodiment 1.
  • the sample AFP concentration is calculated, and multiplied by the dilution factor, which is the total AFP concentration of the sample.
  • the dilution factor which is the total AFP concentration of the sample.
  • detection sub-areas per chip including healthy serum samples, liver cancer serum samples, blank controls, specific chip number and detection sub-area number in Table 1.
  • AFP-L3 index AFP-L3/AFP
  • the current AFP detection level is defined by 20 ng/ml, and normal people are below 20 ng/ml.
  • AFP-L3 (%) > 10 to 15% is a positive judgment indicator.
  • the AFP-L3/AFP ratio of 4 samples encountered in the test was greater than 1, because the AFP concentration of the sample was too high, far exceeding 169 ng/ml. Exceeding the upper limit of the pixel analysis of the chip 255.
  • the test serum can be double-diluted with high-concentration AFP serum and then detected to the actual AFP concentration of the serum.

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Abstract

检测血清糖蛋白岩藻糖指数的化学发光蛋白芯片、试剂盒及检测方法，属于蛋白检测技术。其特征在于：所述蛋白芯片的基质载片上至少包括一个检测亚区，一个所述检测亚区检测一份血清样品；所检测亚区内设置有2个检测斑区域和1个排对照斑区域，其中一个检测斑区域有固定甲胎蛋白的特异性抗体形成的检测斑，另一个检测斑区域有固定小扁豆素形成的检测斑，所述对照斑区域有固定牛血清白蛋白形成的对照斑；同一个检测斑区域内的所有检测斑上的物质的浓度相同。所述蛋白芯片，试剂盒和方法能够准确、高通量地检测血清糖蛋白岩藻糖指数，临床使用中，具有灵敏度高、省时、便捷、经济等优点。

Description

检测血清糖蛋白岩藻糖指数的化学发光蛋白芯片、试剂盒及检测方法 技术领域
本发明涉及蛋白检测技术，特别涉及一种检测血清糖蛋白岩藻糖指数的化学发光蛋白芯片及检测方法。
背景技术
原发性肝癌与肝炎、肝硬变等良性肝病产生的甲胎蛋白(alpha fetoprotein,AFP)在其糖链结构上存在很大差异，即与良性肝病相比，肝癌产生的AFP，其岩藻糖指数要高得多。岩藻糖具有与小扁豆素结合的特性。AFP根据其(岩藻糖基)对小扁豆凝集素的亲和力不同可以分为AFP-L1、AFP-L2和AFP-L3。其中AFP-L1主要来自于良性肝病，AFP-L2主要来源于孕妇，而AFP–L3是甲胎蛋白的岩藻糖糖基化形式，主要来源于HCC。2005年FDA正式批准将AFP-L3作为原发性肝癌的标志物之一。AFP-L3在肝癌的早期诊断、鉴别诊断、疗效评估和预后监测等方面均具有较高的特异性与敏感性。
岩藻糖为甲基化六碳糖，存在于组织及血清多种糖蛋白糖链中，称为蛋白结合岩藻糖(protein-bound fucose,P-bf)。AFP碳水化合物链上存在岩藻糖残基，此种异质体称为岩藻糖基化AFP(FucAFP)，其占AFP总量的百分率称为岩藻糖基化指数(Fucosylation Index,Fuol)。岩藻糖基化指数具有重要的理论意义和临床应用意义，在肝癌诊断和预后应用中可以作为一项重要的指标。
传统的血清岩藻糖蛋白分离方法，包括亲和免疫交叉电泳技术，亲和印迹法，亲和层析法、“双位点夹心”酶联免疫吸附法、LiBASys测定仪、
Figure PCTCN2015084356-appb-000001
i30检测系统技术及热景生物糖基捕获离心柱前处理技术。其中植物凝集素亲和免疫电泳技术和
Figure PCTCN2015084356-appb-000002
i30检测系统技术要求高、操作繁琐、试剂昂贵，限制了其推广应用。而糖基捕获离心柱，由于样本处理和检测分开进行，增加了操作的繁琐性。
发明内容
本发明基于本领域在血清中AFP与AFP-L3的定量检测技术的需求及空白，提供了一种适合于定量检测生物样品中甲胎蛋白和/或岩藻糖基化甲胎蛋白的试剂盒及检测方法，该方案 不仅仅适合于检测血清中的AFP抗原，对检测其他岩藻糖基化蛋白具有通用性，具有省时、经济、准确、便捷的优点。
本发明的技术方案如下：
一种检测血清糖蛋白岩藻糖指数的化学发光蛋白芯片，其特征在于：所述蛋白芯片的基质载片上至少包括一个检测亚区，一个所述检测亚区检测一份血清样品；
所检测亚区内设置有2个检测斑区域和1个排对照斑区域，其中一个检测斑区域有固定甲胎蛋白的特异性抗体形成的检测斑，另一个检测斑区域有固定小扁豆素形成的检测斑，所述对照斑区域有固定牛血清白蛋白形成的对照斑；
同一个检测斑区域内的所有检测斑上的物质的浓度相同。
所述一个检测斑区域至少包括两个所述检测斑。
所述甲胎蛋白的特异性抗体为鼠抗人甲胎蛋白抗体。
所述基质载片上具有多个所述检测亚区，所述每个检测斑区域包括排列成1排的4个检测斑，所述对照斑区域包括排列成1排的4个对照斑；所述检测斑和对照斑排成平行的三列。
所述检测亚区之间设置有凸起作为物理隔断。
一种检测血清糖蛋白岩藻糖指数的化学发光试剂盒，其特征在于：包括权利要求1～4任一所述的化学发光蛋白芯片。
还包括AFP标准品，生物素标记的AFP多克隆抗体，亲和素HRP和HRP化学发光底物液；所述生物素标记的AFP多克隆抗体为兔源抗体，与所述检测斑上固定的AFP特异性抗体来源于不同物种。
还包括用于洗涤和稀释的常规试剂PBST和PBS。
上述任一试剂盒在检测甲胎蛋白和/或岩藻糖基化甲胎蛋白和/或血清糖蛋白岩藻糖指数方面的应用。
一种定量检测岩藻糖基化蛋白的方法，其特征在于：采用上述任一化学发光蛋白芯片，包括如下步骤：
(1)样品检测
将待测血清样本稀释后滴加在所述化学发光蛋白芯片的检测亚区上，孵育后，用PBST洗涤检测亚区，去除非特异结合物；
加入用PBS稀释的生物素标记的AFP抗体,孵育后，用PBST洗涤，去除非特异结合物；
加入PBS稀释的亲和素HRP,孵育后，用PBST洗涤，去除非特异结合物。
加入HRP底物发光液，用化学发光扫描仪对蛋白芯片进行扫描，分别得到稀释后待测血 清样本中的甲胎蛋白的发光像素值以及岩藻糖基化蛋白的发光像素值；
(2)获取甲胎蛋白标准曲线方程及岩藻糖基化蛋白的标准曲线方程：
所述甲胎蛋白标准曲线方程的横坐标x为AFP标准品的梯度浓度值；其纵坐标y为以剃度浓度的AFP标准品为系列待测样品，以步骤(1)的方法检测得到的甲胎蛋白的系列发光像素值；
所述岩藻糖基化蛋白标准曲线方程的横坐标x为AFP-L3标准品中AFP-L3的梯度浓度值；其纵坐标y为以剃度浓度的AFP-L3标准品为系列待测样品，以步骤(1)的方法检测得到的岩藻糖基化蛋白的系列发光像素值；所述AFP-L3标准品为含有岩藻糖基蛋白(AFP)的血清；
(3)步骤(1)中的待测血清样品的甲胎蛋白的发光像素值带入所述甲胎蛋白标准曲线方程中计算得到稀释后血清的甲胎蛋白浓度，乘以稀释倍数得到待测血清甲胎蛋白浓度；步骤(1)中的待测血清样品的岩藻糖基化蛋白的发光像素值带入所述岩藻糖基化蛋白标准曲线方程中计算得到稀释后的血清的岩藻糖基化蛋白浓度，乘以稀释倍数得到待测血清岩藻糖基化蛋白的浓度；
待测血清岩藻糖基化蛋白的浓度与所述待测血清甲胎蛋白浓度的比值岩藻糖基化指数。
所述孵育指37℃孵育30分钟。
本发明提供一种检测血清糖蛋白岩藻糖指数的化学发光蛋白芯片，基于抗体抗原抗体夹心反应原理及化学发光原理，同时固定有甲胎蛋白特异性抗体和小扁豆素，甲胎蛋白特异性抗体用于结合血清中的所有甲胎蛋白(AFP-L1、AFP-L2和AFP-L3)，小扁豆素用于结合岩藻糖基化甲胎蛋白。同时设置有对照斑点。可以在绝对相同的条件下同时检测到待测血清中甲胎蛋白总浓度以及岩藻糖基化甲胎蛋白的浓度，准确地获得血清糖蛋白岩藻糖指数。本发明提供的化学发光蛋白芯片至少包括一个亚检测区，可以检测一个血样。在大部分实施例中，优选设置至少两个检测亚区，其中一个亚区用于检测对照血清，另一个亚区用于检测待测血样。进一步地，为了实现高通量检测，优选为多个，例如，三个，四个，五个，六个，七个，八个，九个或十个检测亚区，这样就可以在一张芯片上检测多个血清样品，提高临床检测效率，降低成本。如图1所示，本发明的一个优选实施例中，一个所述检测亚区内包括4个固定有AFP特异性抗体的检测斑，4个固定有小扁豆素的检测斑和4个对照斑；两种检测斑和对照斑各排成平行的三列。
本发明还提供了一种检测血清糖蛋白岩藻糖指数的化学发光试剂盒，其中包括上述蛋白芯片以及化学发光的常规试剂，标准曲线方程数据等。
本发明蛋白质芯片的使用有三方面的优势：
一、在基本上完全相同的条件下检测岩血清中的甲胎蛋白和藻糖基化甲胎蛋白，以保证测得的岩藻糖基化指数更准确可靠。
二、允许同时检测多个样品。多个重复的样品，或者不同时间点取的样品以获得动态值，或者各个不同的样品，总之，实现高通量检测。整体上降低检测成本和提高检测效率。
三、采用本发明的蛋白质芯片需要的血样及抗体量都大为减少，仅需原始血清量2.5ul～10ul,而ELISA方法检测需血清50ul；蛋白芯片板抗体点样，5ul至少可以点样20张芯片，检测200份血清,抗体需要量远远低于ELISA方法，大大降低了检测成本和费用。
同时，本发明还提供了利用所述试剂盒对岩藻糖基化甲胎蛋白进行定量检测的方法。首先本发明采用购买的AFP抗原标准品，制成梯度浓度的待测AFP稀释液，通过化学发光检测方法测定每个梯度对应的发光像素值，以梯度浓度为横坐标，荧光像素值为纵坐标制定标准曲线并得到直线回归方程。
本发明提供的检测血清糖蛋白岩藻糖指数的方法，是在上述蛋白芯片上，利用抗体和抗原特异性结合，小扁豆素特异性结合的特点，加入血清或者血浆标本进行孵育，然后加入生物素标记的AFP多克隆抗体，HRP标记的亲和素，最后加入HRP发光底物，通过化学发光扫描仪对发光信号进行扫描量化。将获得的信号值带入预先制作好的直线回归方程中，得到样品中岩藻糖蛋白AFP-L3的浓度。
本发明的方法的检测原理与一般化学发光免疫反应有所区别，通常的化学发光免疫反应Elisa反应形成“抗体－抗原－辣根过氧化物酶标记的二抗”复合物，最后加入HRP化学发光底物液获得发光值。但是，由于辣根过氧化物酶本身是存在糖残基，如果在本发明中的二抗采用辣根过氧化物酶标记，辣根过氧化物酶的糖残基会结合小扁豆素，从而严重干扰检测值，本发明做过的一些实验证明，这样得不到准确的岩藻糖基化指数，假阳性非常高，正常血清都能测出很高的岩藻糖基化指数。基于此，本发明提供的芯片及方法的原理如下：将抗AFP单克隆抗体和小扁豆素有序的固定在蛋白芯片上，依次加入待测血清、生物素标记的AFP多克隆抗体和亲和素HRP,分别形成“AFP单抗－AFP－生物素标记AFP多抗－亲和素HRP复合物”，以及“小扁豆素－AFP-L3－生物素标记的AFP抗体－亲和素HRP复合物”，最后加入HRP化学发光底物液进行孵育，利用化学发光仪进行扫描得到发光像素值，将像素值代入标准曲线对应的直线回归方程式可分别得出AFP和AFP-L3的浓度，从而获得岩藻糖基化甲胎蛋白AFP-L3占AFP总量的百分率，即岩藻糖指数。
实验结果证明，本发明的方法不仅可以进行定性检测，还可通过发光强度对AFP和岩藻糖基化AFP进行定量检测。与ELISA方法进行对比，敏感性和特异性均优于ELISA方法， 从时间上比较，ELISA检测至少需要3小时，本发明仅需要1.5小时；从抗体用量上比较，利用办发明的试剂盒中的蛋白质芯片进行抗体点样，5ul抗体至少可以点样20张芯片，检测200份血清,抗体需要量远远低于ELISA方法；从血清用量上比较，ELISA方法检测需血清50ul，而本发明的试剂盒和检测方法检测一份血清样品只需要2.5ul～10ul的原始血清量；因此，本发明提供的试剂盒和检测方法具有灵敏度高、省时、经济等特点，可以大大地降低血液蛋白检测的成本和时间。
综上，本发明的方法结合了化学发光检测方法、标准曲线以及蛋白芯片技术的应用，确保了利用试剂盒进行AFP-L3定量检测结果的高灵敏性，准确性、高效性和低成本。本发明提供的检测方法一种可行、可靠、经济，且简单、省时的方法。本发明的技术方案将为大规模、高通量检测血清中岩藻糖基化甲胎蛋白提供了一种经济、可靠的试剂盒和检测方法。
附图说明
图1.AFP/扁豆素蛋白质芯片点样示意图；
图2.AFP/扁豆素抗体夹心法蛋白质芯片流程图；
图3.AFP蛋白质芯片检测AFP标准品结果扫描图
蛋白芯片的点样抗体为不同浓度的AFP抗体，A:1mg/ml；B:0.5mg/ml；C:0.25mg/ml；检测物分别为1.80ng/ml，2.40ng/ml，3.20ng/ml，4.10ng/ml，5.5ng/ml，6.肝癌血清，7.肝癌血清，8.空白对照，9.健康血清，10.肝癌血清.
图4.AFP蛋白质芯片检测AFP3标准曲线图及回归方程a.
图5.AFP蛋白质芯片检测AFP标准品和血清样本扫描图。
检测物分别为，(1-5)80ng/ml,40ng/ml，20ng/ml，10ng/ml，5ng/ml，6.肝癌血清，7.肝癌血清，8肝癌血清，9肝癌血清，10肝癌血清(芯片抗体点样抗体为AFP 0.5mg/ml)
图6.AFP/扁豆素点样芯片,检测AFP-L3标准品结果扫描图
A:AFP抗体0.5mg/ml；B:小扁豆素4mg/ml；点样不同浓度的血清浓度的AFP-L3：(1-5)100ng/ml；50ng/ml；25ng/ml；12.5ng/ml；6.25ng/ml；(6-9)100ng/ml；50ng/ml；25ng/ml；12.5ng/ml.(10)空白对照。
图7.AFP/扁豆素点样芯片检测AFP-L3标准曲线图及回归方程b.
图8.AFP/扁豆素点样芯片检测肝癌和正常血清样本扫描图.
8张芯片，检测39份肝癌血清样本，32份正常健康血清样本，9份空白对照。
具体实施方式
下面结合具体实施方式对本发明作进一步的详细说明，但并不限制本发明的范围。如无特殊说明，下述实施例中使用的操作均为常规方法，所采用的试剂均可以商购获得。
主要仪器设备
化学发光扫描仪，由军事医学科学院研制。
主要试剂及其来源
鼠源单克隆抗体AFP(深圳菲鹏公司)、小扁豆素(Sigma公司)、醛基芯片(上海百傲公司)、生物素标记的兔源抗体、HRP标记的亲和素(美国abcam公司)、HRP化学发光底物液A液和B液，按照1：1比例混合，新鲜配置。(美国Millipore公司)。
实施例1.蛋白质芯片制备及使用流程
实验所用试剂和仪器：鼠源单克隆抗体AFP(深圳菲鹏公司)；小扁豆素(Sigma公司)；醛基芯片(上海百傲公司)；生物素标记的兔源一抗(美国abcam公司)；亲和素HRP(美国abcam公司),化学发光扫描仪。(军事医学科学院王升启教授实验室研制)
PBS配方:氯化钠(NaCl)8g，氯化钾(KCl)0.2g，磷酸氢二钠(Na2HPO4)1.44g，磷酸二氢钾(KH2PO4)0.24g，调pH 7.4,定容1L
PBST配方：PBS,1L+Tween-20,1ml
芯片为醛基芯片(上海百傲公司)，每张芯片包含10个检测方格(检测亚区)，每个方格检测一份血清，一次检测10份血清。
每个检测方格内，将鼠源单克隆抗体AFP(深圳菲鹏公司)、小扁豆素Sigma公司)依次点在芯片上，点样四次，点样浓度单克隆抗体AFP 0.5mg/ml，小扁豆素4mg/ml，点成两排八个检测斑；10％牛血清白蛋白(BSA)作为阴性对照，同样点样四次，点成对照斑
蛋白芯片操作流程：
用制备好的蛋白质芯片检测健康对照组和肝癌实验组动态血清标本中的肿瘤标志物。
用血清样本10ul,(或者2.5ul稀释4倍),滴加在芯片上，37℃孵育30分钟，利用抗原抗体相结合的特性，以及小扁豆素和岩藻糖结合的特性，使血清中的AFP与芯片上相对应的(鼠源)抗体特异性结合，形成抗原抗体(鼠源)复合物；小扁豆素与岩藻糖结合形成小扁豆素抗原复合物。
用PBST洗涤4次，去除非特异结合，再加入PBS稀释的生物素标记兔源一抗，37℃孵育30分钟。兔源抗体与抗原结合，形成鼠源抗体-AFP-兔源生物素标记抗体复合物,和小扁豆素-岩藻糖基化AFP-兔源生物素标记抗体复合物。
用PBST洗涤4次，去除非特异结合，再加入PBS稀释的亲和素HRP，37℃孵育30分钟。生物素与亲和素结合，形成“鼠源抗体-AFP-兔源生物素标记抗体-亲和素HRP复合物”,和“小扁豆素-岩藻糖基化AFP-兔源生物素标记抗体-亲和素HRP复合物”。
用PBST洗涤4次，去除非特异结合，加入HRP发光底物，37℃孵育30分钟，化学发光扫描仪进行扫描。
固相载体上的化学发光像素与标本中受检抗原的量成正向相关,此时测定复合物中的像素值，即可确定待测抗原含量。芯片点样抗体(鼠源一抗)和检测用的抗体(兔源一抗)分别取自不同种属的动物。如图示2抗体夹心法蛋白芯片流程图。
实施例2.本发明检测方法的建立
(1)标准曲线及回归方程式a.
采用购买的AFP抗原(美国abcam公司)，设置成不同的浓度梯度，(1-5)80ng/ml,40ng/ml，20ng/ml，10ng/ml，5ng/ml，6.肝癌血清，7.肝癌血清，8空白对照，9健康血清，10肝癌血清(图3，芯片抗体点样抗体为AFP A:1mg/ml；B:0.5mg/ml；C:0.25mg/ml)。
采用实施例1中的操作流程和蛋白质芯片检测AFP标准品各个浓度梯度，检测扫描结果见图3。检测结果绘制成标准曲线图，以标准物的浓度为横坐标，像素值为纵坐标，在坐标纸上绘出标准曲线。根据样品的像素值由标准曲线查出相应的浓度；再乘以稀释倍数；或用标准物的浓度与OD值计算出标准曲线的直线回归方程式，将样品的OD值代入方程式，计算出样品浓度，再乘以稀释倍数，即为样品的实际浓度，标准曲线及回归方程a见图4。
采用购买的AFP抗原(美国abcam公司)，设置成不同的浓度梯度，(1-5)80ng/ml,40ng/ml，20ng/ml，10ng/ml，5ng/ml，6.肝癌血清，7.肝癌血清，8肝癌血清，9肝癌血清，10肝癌血清(图5，芯片抗体点样抗体为AFP 0.5mg/ml)。
采用实施例1中的操作流程和蛋白质芯片检测AFP标准品各个浓度梯度，检测扫描结果见图3。检测结果绘制成标准曲线图，以标准物的浓度为横坐标，像素值为纵坐标，在坐标纸上绘出标准曲线。根据样品的像素值由标准曲线查出相应的浓度；再乘以稀释倍数；或用标 准物的浓度与OD值计算出标准曲线的直线回归方程式，将样品的OD值代入方程式，计算出样品浓度，再乘以稀释倍数，即为样品的实际浓度，标准曲线及回归方程a见图4。
(2)标准曲线及回归方程式b.
采用已知AFP-L3浓度的血清，倍比稀释，设置成不同的浓度梯度，(1-5)200ng/ml,100ng/ml，50ng/ml，25ng/ml，12.5ng/ml，(6-9)200ng/ml,100ng/ml，50ng/ml，25ng/ml.10空白对照(图6)。
采用实施例1中的操作流程和蛋白质芯片检测血清(AFP-L3)标准品各个浓度梯度，检测扫描结果见图6。检测结果绘制成标准曲线图，以标准物的浓度为横坐标，像素值为纵坐标，在坐标纸上绘出标准曲线。根据样品的像素值由标准曲线查出相应的浓度；再乘以稀释倍数；或用标准物的浓度与OD值计算出标准曲线的直线回归方程式，将样品的OD值代入方程式，计算出样品浓度，再乘以稀释倍数，即为样品的实际浓度，标准曲线及回归方程b见图7。
实施例3.样品检测检验本发明方法的稳定性，准确性和可靠性
血清样本：
39份肝癌血清：来源于首都医科大学附属佑安医院标本库；
32份正常健康人血清；
9份空白对照(空白对照为1×PBS)。
检测流程同实施例1.
将样品的像素值代入图4的回归方程式a，计算出样品AFP浓度，再乘以稀释倍数，即为样品的AFP总浓度。将样品的像素值代入图7的回归方程式b，计算出样品AFP-L3浓度，再乘以稀释倍数，即为样品的AFP-L3总浓度。
每张芯片10个检测亚区，包括健康血清样本，肝癌血清样本，空白对照，具体分表1中芯片编号及检测亚区编号。
样品检测结果扫描结果见图8。
计算公式：AFP总浓度X＝{(扫描像素值Y-39.05)/5.476}×稀释倍数。
AFF-l3总浓度X＝{(扫描像素值Y-24.65)/2.26}×稀释倍数
AFP-L3指数＝AFP-L3/AFP
检测结果如下表1.
表1 临床样本检测结果汇总表。
Figure PCTCN2015084356-appb-000003
Figure PCTCN2015084356-appb-000004
表2. 80血清的AFP-L3/AFP
Figure PCTCN2015084356-appb-000005
目前的AFP检测水平以20ng/ml为分界线，正常人低于20ng/ml。AFP-L3(％)>10～15％为阳性判断指标。
本芯片的检测结果：
空白对照9份没有检测到AFP和AFP-L3：说明本实验采用的芯片有效。
健康血清32份，没有检测到AFP和AFP-L3；说明本发明提供的芯片及方法检测的假阳性为0。
39份肝癌血清中37份检测到AFP(94.87％)，其中35份肝癌血清标本的AFP含量高于20ng/ml(89.74％)；26份肝癌血清中检测到AFP和AFP-L3，2份样本既没有检测到AFP,也没有检测到AFP-L3。在检测到AFP和AFP-L3的26份肝癌血清样本中22份的AFP-L3/AFP比例大于10％(84.61％)，4份样本的AFP-L3/AFP比例小于10％。证明：本发明的芯片和方法具有35/39＝89.74％检出灵敏性，100％的特异性，具有可靠的临床应用价值。
上述数据说明，本发明的芯片和方法稳定性，准确性和可靠性良好
检测中遇到4份样本的AFP-L3/AFP比例大于1，是因为样品的AFP浓度过高，远远超过了169ng/ml。超出了该芯片像素分析的上限值255.遇到这种情况时，检测血清可通过加倍稀释高浓度AFP血清,再检测，到该血清的实际AFP浓度。

Claims (11)

  1. 一种检测血清糖蛋白岩藻糖指数的化学发光蛋白芯片，其特征在于：所述蛋白芯片的基质载片上至少包括一个检测亚区，一个所述检测亚区检测一份血清样品；
    所检测亚区内设置有2个检测斑区域和1个排对照斑区域，其中一个检测斑区域有固定甲胎蛋白的特异性抗体形成的检测斑，另一个检测斑区域有固定小扁豆素形成的检测斑，所述对照斑区域有固定牛血清白蛋白形成的对照斑；
    同一个检测斑区域内的所有检测斑上的物质的浓度相同。
  2. 根据权利要求1所述的化学发光蛋白芯片，其特征在于：所述一个检测斑区域至少包括两个所述检测斑。
  3. 根据权利要求1所述的化学发光蛋白芯片，其特征在于：所述甲胎蛋白的特异性抗体为鼠抗人甲胎蛋白抗体。
  4. 根据权利要求1所述的化学发光蛋白芯片，其特征在于：所述基质载片上具有多个所述检测亚区，所述每个检测斑区域包括排列成1排的4个检测斑，所述对照斑区域包括排列成1排的4个对照斑；所述检测斑和对照斑排成平行的三列。
  5. 根据权利要求4所述的化学发光蛋白芯片，其特征在于：所述检测亚区之间设置有凸起作为物理隔断。
  6. 一种检测血清糖蛋白岩藻糖指数的化学发光试剂盒，其特征在于：包括权利要求1～4任一所述的化学发光蛋白芯片。
  7. 根据权利要求6所述的化学发光试剂盒，其特征在于：还包括AFP标准品，生物素标记的AFP多克隆抗体，亲和素HRP和HRP化学发光底物液；所述生物素标记的AFP多克隆抗体为兔源抗体，与所述检测斑上固定的AFP特异性抗体来源于不同物种。
  8. 根据权利要求6所述的化学发光试剂盒，其特征在于：还包括用于洗涤和稀释的常规试剂PBST和PBS。
  9. 权利要求6-8任一所述的试剂盒在检测甲胎蛋白和/或岩藻糖基化甲胎蛋白和/或血清糖蛋白岩藻糖指数方面的应用。
  10. 一种定量检测岩藻糖基化蛋白的方法，其特征在于：采用权利要求1～5任一所述的化学发光蛋白芯片，包括如下步骤：
    (1)样品检测
    将待测血清样本稀释后滴加在所述化学发光蛋白芯片的检测亚区上，孵育，然后用PBST 洗涤检测亚区，去除非特异结合物；
    加入用PBS稀释的生物素标记的AFP抗体,孵育，然后用PBST洗涤，去除非特异结合物；
    加入PBS稀释的亲和素HRP，孵育，然后用PBST洗涤，去除非特异结合物；
    加入HRP底物发光液，用化学发光扫描仪对蛋白芯片进行扫描，分别得到稀释后待测血清样本中的甲胎蛋白的发光像素值以及岩藻糖基化蛋白的发光像素值；
    (2)获取甲胎蛋白标准曲线方程及岩藻糖基化蛋白的标准曲线方程：
    所述甲胎蛋白标准曲线方程的横坐标x为AFP标准品的梯度浓度值；其纵坐标y为以剃度浓度的AFP标准品为系列待测样品，以步骤(1)的方法检测得到的甲胎蛋白的发光像素值；
    所述岩藻糖基化蛋白标准曲线方程的横坐标x为AFP-L3标准品中AFP-L3的梯度浓度值；其纵坐标y为以剃度浓度的AFP-L3标准品为系列待测样品，以步骤(1)的方法检测得到的岩藻糖基化蛋白的发光像素值；所述AFP-L3标准品为含有岩藻糖基蛋白(AFP)的血清；
    (3)步骤(1)中的待测血清样品的甲胎蛋白的发光像素值带入所述甲胎蛋白标准曲线方程中计算得到稀释后血清的甲胎蛋白浓度，乘以稀释倍数得到待测血清甲胎蛋白浓度；步骤(1)中的待测血清样品的岩藻糖基化蛋白的发光像素值带入所述岩藻糖基化蛋白标准曲线方程中计算得到稀释后的血清的岩藻糖基化蛋白浓度，乘以稀释倍数得到待测血清岩藻糖基化蛋白的浓度；
    待测血清岩藻糖基化蛋白的浓度与所述待测血清甲胎蛋白浓度的比值岩藻糖基化指数。
  11. 根据权利要求10所述的方法，所述孵育指37℃孵育30分钟。
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