WO2017008389A1 - 胰腺癌蛋白生物标记物的检测试剂盒及检测系统 - Google Patents

胰腺癌蛋白生物标记物的检测试剂盒及检测系统 Download PDF

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WO2017008389A1
WO2017008389A1 PCT/CN2015/089895 CN2015089895W WO2017008389A1 WO 2017008389 A1 WO2017008389 A1 WO 2017008389A1 CN 2015089895 W CN2015089895 W CN 2015089895W WO 2017008389 A1 WO2017008389 A1 WO 2017008389A1
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monoclonal antibody
kit
pancreatic cancer
antibody
detecting
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PCT/CN2015/089895
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English (en)
French (fr)
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张贯京
克里斯基捏 普拉纽克
艾琳娜 古列莎
伊万 波达别特
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深圳市华科安测信息技术有限公司
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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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • 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
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification
    • G01N2800/7023—(Hyper)proliferation
    • G01N2800/7028—Cancer

Definitions

  • the invention belongs to the technical field of biological detection, and particularly relates to a detection kit and a detection system for a pancreatic cancer protein biomarker.
  • Pancreatic cancer is a malignant tumor of the digestive tract with a high degree of malignancy and is difficult to diagnose and treat. About 90% of ductal adenocarcinomas originate from the ductal epithelium. Usually after onset, the 5-year survival rate is ⁇ 1%, which is one of the worst prognosis.
  • pancreatic cancer The important reason for the low prognosis of pancreatic cancer is that there is no symptom in the early stage of pancreatic cancer, and it is difficult to detect it during the incubation period or initial stage of pancreatic cancer, thus delaying the optimal treatment time. It is mainly difficult to be detected because there is no symptom at the beginning of the disease, and there is a certain positive rate when there is no detection of CA19-9, CEA, CA242 pancreatic cancer detection markers with the best detection accuracy. High specificity. According to statistics, the use of the above markers in the detection of pancreatic cancer during pregnancy (before canceration) is less sensitive, such as 1 year before the onset, 68% sensitivity, two years before the onset, the sensitivity is 53%. The use of other routine clinical techniques, such as B-ultrasound, CT, MRI, ERCP, PTCD, angiography, laparoscopic examination, etc., found that cancer has been in the advanced stage of cancer, greatly delaying the diagnosis and treatment.
  • the object of the present invention is to overcome the above-mentioned deficiencies of the prior art and to provide a detection kit for pancreatic cancer protein biomarkers which can be more accurate and specific.
  • a kit for detecting pancreatic cancer protein biomarkers comprising CRP monoclonal antibody, ICAM-1 monoclonal antibody, OPG monoclonal antibody and CA19-9 monoclonal antibody, and an enzyme-labeled secondary antibody for forming a double-antibody sandwich.
  • the present invention further provides a detection system comprising the above-described pancreatic cancer protein biomarker detection kit.
  • the detection kit and the detection system of the present invention screen the obtained pancreatic cancer protein biomarkers of CRP, ICAM-1, OPG and CA19-9 by means of "differential subtraction" for the present invention, and perform immunological preparation specificity.
  • CRP monoclonal antibody, ICAM-1 monoclonal antibody, OPG mAb and CA19-9 mAb Based on CRP monoclonal antibody, ICAM-1 monoclonal antibody, OPG mAb and CA19-9 mAb, and binding to the double-antibody sandwiched secondary antibody, specific binding by antigen-antibody and combined with the secondary antibody against the sandwich
  • the detection of the above biomarker proteins has relatively high accuracy and specificity.
  • FIG. 1 is a schematic view showing the arrangement of spotting holes on a solid phase substrate in a chip according to an embodiment of the present invention
  • FIG. 2 is a schematic view showing the overall shape of a pancreatic cancer protein biomarker after the detection chip is packaged according to an embodiment of the present invention.
  • the present invention provides a pancreatic cancer protein biomarker comprising CRP (C-reactive protein), ICAM-1 (intercellular adhesion molecule-1), OPG (osteoprotegerin) and CA19-9 (sugar chain antigen 19-9) .
  • the present invention proposes the above biomarkers based on the detection and examination of pancreatic cancer, and the markers of the present invention are more accurate than the conventional clinical analysis means and the commonly used CA19-9, CEA, CA242 pancreatic cancer detection markers.
  • the discovery and presentation of the above-described combination of biomarkers of the present invention is based on the fact that the biomarker itself is a measurable indicator reflecting the presence and state of the disease, and any physiological changes in the course of disease, stress and recovery can ultimately It is the most intuitive means of disease research at the protein level; and multiple protein biomarker groups are closely related to the clinical and prognosis of patients with chronic diseases, indicating that simultaneous detection of multiple protein factors contributes to a more comprehensive understanding of the disease. process.
  • the protein in the above-mentioned biomarker combination belongs to a regulatory protein peculiar to a disease. Because tissue fluids, such as plasma, have thousands of protein species and concentration ranges up to 1010, the most advanced instruments can only measure a concentration range of 103; to overcome the interference of healthy background proteins on disease-specific protein.
  • the above marker proteins were screened by differential analysis and verified. The specific screening process is as follows:
  • a total of 210 subjects with known physical conditions were selected, including 55 healthy controls, 65 patients with initial pancreatic cancer (not metastasized), and 90 patients with metastatic pancreatic cancer. (This study has been approved by an ethics review agency and all subjects have signed informed consent.)
  • the most advanced instruments can only measure the concentration range span 103; currently, antibodies are used to remove 20 to 100 high-abundance proteins in the sample.
  • the detection rate of low-abundance proteins is increased, when compared with more than 1000 proteins in plasma, the residual relatively high-abundance protein has a ng concentration on the markers used in cancer and cancer-related developmental gestation-related markers.
  • the analysis signal of the pg concentration still has a severe flooding effect.
  • the present invention adopts a subtractive subtraction technique, and uses a healthy control plasma sample as an antigen to immunize a rabbit, and prepares a polyclonal antibody against a healthy control plasma protein, and uses the antibody as an affinity adsorption medium to prepare a chromatography column.
  • a large number of health-related proteins in patient plasma samples are specifically adsorbed and removed to obtain as much disease-related as possible, excluding interference from health-related high-abundance proteins.
  • low-abundance differentially expressed proteins were analyzed and detected by SDS-PAGE and mass spectrometry after removing a large amount of background interference from high-abundance proteins and enriching the amount.
  • the healthy control group In the early screening stage, the healthy control group, the initial stage of pancreatic cancer (not metastasized), and the patients with metastatic pancreatic cancer were identified by mass spectrometry, and the protein was identified three times in each of the three replicates of each group, and 210, 320, and 378 proteins were identified. protein.
  • the present invention aims to find early biomarkers of pancreatic cancer, which are only found in patients with initial pancreatic cancer (untransferred) and metastatic pancreatic cancer, but there are 45 proteins that are not present in healthy controls. There were 179 proteins in healthy controls, early pancreatic cancer patients (not metastasized), and patients with metastatic pancreatic cancer. This trial is aimed at the analysis of only 45 proteins co-occurring in patients with early stage pancreatic cancer (no metastasis) and metastatic pancreatic cancer.
  • CRP C-reactive protein
  • ICAM-1 intercellular adhesion molecule-1
  • OPG osteoprotegerin
  • CA19-9 CA19-9 in the pancreatic cancer group.
  • the amount is significantly higher than the healthy control and increases as the patient's condition is severe (increased proteinuria).
  • the relative content of these four protein markers was determined by reference to the normal range and the cutoff values of the four protein molecules to determine the relative content of the four protein markers.
  • the various combinations were statistically processed to compare the sensitivity and specificity of the different combinations. The results are shown in Table 3 below. Among them, in the combination indicated by the symbol " ⁇ " in Table 3, sensitivity and specificity were calculated when all the biomarkers contained in the combination were positive (i.e., higher than the cutoff value). In the combination indicated by the symbol " ⁇ ", when one of the biomarkers contained in the combination is positive, it is judged to be positive, and the sensitivity and specificity are calculated.
  • the invention further provides a detection chip for a pancreatic cancer protein biomarker based on the combination of the above pancreatic cancer protein biomarker, the detection chip comprises: a solid phase carrier, and a CRP monoclonal antibody immobilized on the solid phase carrier, ICAM- 1 mAb, OPG mAb and CA19-9 mAb.
  • the detection chip of the pancreatic cancer protein biomarker of the present invention by immobilizing the monoclonal antibody of the above protein biomarker on a solid phase carrier, and capturing the protein to be tested which can specifically bind thereto by the corresponding monoclonal antibody, thereby realizing Separation of the target marker protein in serum, plasma, lymph, interstitial fluid, urine, exudate, cell lysate, and secretion sample waiting for the sample; followed by washing, purification, confirmation, and biochemical analysis; The differential expression results of these biomarker proteins in the sample to be tested are obtained.
  • the monoclonal antibody of the four biomarker proteins contained in the detection chip of the pancreatic cancer protein biomarker is used in the present invention to immunize a monoclonal antibody by using the rabbit as an immunogen, for example, the preparation of CRP monoclonal antibody is Examples include:
  • CRP protein was used as an antigen to enter the peripheral immune organs of rabbits through blood circulation or lymphatic circulation, and the corresponding B lymphocyte clones were stimulated and activated. Proliferate and differentiate into sensitized B lymphocytes;
  • the rabbit When the immunization reaches the target level, the rabbit is sacrificed, and the spleen of the rabbit is taken; after the fine crushing, the single cell suspension is digested by enzymatic digestion, and the cell fusion hybridization is performed with the tumor cells;
  • the successful fusion cell dilution method is used for cloning and culture of hybrid cells; after culture, positive hybridoma cells capable of producing monoclonal antibodies against CRP protein are screened by immunization method; and the positive hybridoma cells are cultured in vitro. In the process of culture, the expression is induced at the appropriate time, and after the completion of the culture, the CRP monoclonal antibody can be obtained.
  • CRP monoclonal antibody, ICAM-1 monoclonal antibody, OPG monoclonal antibody and CA19-9 monoclonal antibody are separately prepared, and then fixed to a solid phase carrier by covalent crosslinking or non-covalent crosslinking.
  • the solid phase carrier as a protein chip in practice can generally be selected from a nitrocellulose membrane, a nylon membrane, a gold membrane, a flat plate or a glass substrate.
  • a nitrocellulose membrane or a glass substrate is preferably used for the purpose of performing low-abundance differential protein-specific adsorption for the above-mentioned monoclonal antibody. The reason is that the nitrocellulose membrane itself is a permeation membrane, and the nitrocellulose cellulose microporous membrane has a large capacity for an empty structure and a strong binding force to proteins.
  • the use of a glass substrate is mainly convenient, inexpensive, easy to handle, and has sufficient stability and inertness, and is very suitable for large-scale commercial production of the product of the present invention.
  • the glass substrate needs to be treated according to the characteristics of the present invention, because the glass substrate as the protein carrier is a glass plate, and the surface hydroxyl group is firstly used with N,N-diethoxyaminopropyltriethoxylate.
  • the silane is surface-treated; then the antibody protein is coupled, but the glass substrate has a very strong non-specific adsorption, which is likely to cause very serious background interference, especially in the above four kinds of target proteins to be detected in the present invention.
  • the glass substrate is used as a solid phase carrier in the present invention
  • the glass substrate is surface-treated with PEG (polyethylene glycol), and the surface treated with PEG has better signal intensity than the siliconized surface, and the macromolecular analysis component can be improved
  • PEG polyethylene glycol
  • the ability to bind to a ligand, such as a ligand, can attenuate non-specific binding; this is because PEG has a large spatial structure, which reduces the steric hindrance between proteins and can increase the specificity of affinity adsorption.
  • a positive control and a negative control are also immobilized on the above-mentioned protein chip of the present invention.
  • the positive control can use IgG monoclonal antibody, and the positive control IgG monoclonal antibody is preferably prepared by using the same immune body as above.
  • the positive control is prepared by using rabbit as an immunogen, then the IgG monoclonal antibody is also the best. It is prepared by using rabbit as an immunological body; the positive control is used to detect the binding of the target protein.
  • the negative control can use a sample-like dilution of the sample, which does not contain any bindable protein, just as a blank control.
  • the solid phase carrier of the above-mentioned protein chip of the present invention is provided with spotting holes, and the spotting holes may be disposed on the surface of the solid phase carrier in a matrix or array manner.
  • the corresponding monoclonal antibodies used to detect the biomarker protein are then immobilized in these spotted wells.
  • the structure of the sampled hole is used for operation detection.
  • the protein in the sample hole can be well separated, and the cross-contamination is minimized; on the other hand, the concave structure of the hole can facilitate the addition and preservation of the spotting buffer.
  • the rules and sizes of the spotting holes can be standardized, which facilitates horizontal comparison of the amount of spotting. Controlling the amount of sample is also more conducive to lateral comparison and calculation of results.
  • the present invention also provides a detection kit for a pancreatic cancer protein biomarker for the purpose of specifically detecting the above-mentioned pancreatic cancer protein organism for the convenience of the detection chip of the pancreatic cancer protein biomarker described above.
  • the above four monoclonal antibodies are respectively used as a chromatographic medium to form a chromatography column, and then the sample to be tested is subjected to a column, and then assisted by electrophoresis, mass spectrometry and the like.
  • the enzyme-labeled antigen may be used, and the sample protein to be tested is subjected to an enzyme labeling, and then subjected to an affinity reaction with the above-mentioned four monoclonal antibodies, and then the signal is detected by the enzyme-labeled antigen bound to the respective monoclonal antibody.
  • the kit of the present invention refers to the double-anti-sandwich method to assist the auxiliary reagent, and further the auxiliary reagent includes the sample dilution buffer.
  • the sample dilution buffer In the invention, 30 ⁇ 70 mM PBS phosphate buffer containing 1.5% to 2% BSA (bovine serum albumin) stabilizer is used as the sample dilution buffer; the sample dilution buffer is used for sample dilution and washing, wherein BSA is Protein stabilizers prevent protein breakdown and non-specific adsorption; and BSA also mitigates some adverse environmental factors such as heat, surface tension and chemical factors, thereby reducing the denaturation of some proteins during the assay.
  • BSA bovine serum albumin
  • the above kit of the present invention further needs to cooperate with the enzyme-labeled secondary antibody according to the principle of the double-anti-sandwich method; after the sample to be tested, each protein of interest will be It is captured by the corresponding monoclonal antibody and affinity-adsorbed. At this time, an excess of the enzyme-labeled secondary antibody is added to bind to the target protein to form a sandwich structure; then the amount of the enzyme-labeled secondary antibody can be reacted in the sample to be tested.
  • the expression of the target protein can be obtained by calculating the signal carried by the enzyme-labeled secondary antibody.
  • the above-mentioned enzyme-labeled secondary antibody can be carried out by using a relatively common HRP (horseradish peroxidase)-containing enzyme-labeled secondary antibody, and in the case of satisfying the accuracy, the self-designed and manufactured product is also omitted. trouble.
  • HRP horseradish peroxidase
  • the kit may further comprise a concentrated washing solution containing 1 ⁇ 2%. BSA, 0.03 ⁇ 0.07% Tween-20 PBS, used to concentrate the enzyme-labeled secondary antibody and signal detection color components.
  • BSA 0.03 ⁇ 0.07% Tween-20 PBS
  • TMB tetramethylbenzidine
  • a solid phase matrix can also be provided in the kit, which in practice is a matrix similar to the above-mentioned chip, functioning as a carrier and a reaction medium.
  • the specific shape and morphology can also be referred to in the above description of the chip.
  • the corresponding monoclonal antibody is fixed to the spotting hole of the solid phase matrix according to the instructions of the kit manual, and then the detection of the sample to be tested is performed;
  • the solid phase matrix carrier is not provided in the kit of the present invention, it can also be carried out by using a spotting plate of the ELISA kit.
  • kits for the setting of the comparison, refer to the description in the above chip, and the usage is the same as the sample to be tested, and the operation is performed synchronously with the sample to be tested, and then the results are compared to provide a comparative reference.
  • the present invention also proposes a pancreatic cancer detecting system comprising the above-described pancreatic cancer protein biomarker detection kit based on the above detection kit for pancreatic cancer protein biomarkers.
  • the detection system of the present invention is based on the use of the kit, and the direct result can not be directly obtained, so that the detection device device, such as a CCD detector, which is matched with the kit, is combined to be similar; these devices and instruments are combined. It can detect the combined change signal of the reagent in the kit, and finally output the clear visual data result directly to make a whole.
  • the detection system of the invention comprising the detection kit combines the kit product with the equipment that can be used together with the kit, etc., and detects the biomarker protein by specific binding of the antigen-antibody, and finally
  • the intuitive data results that can be directly and completely detected are more convenient, intuitive and accurate.
  • Example 1 is used to demonstrate the accuracy of pancreatic cancer detection using the above-described biomarkers of the present invention and the authenticity of the test results using the kit.
  • a 50 mM pH 7 containing 2% BSA was added dropwise to the spotted well. .2
  • the PBS buffer solution was then incubated with a cover slip for 2 h; the front and back surfaces of the slides blocked with BSA were washed three to four times with 50 mM pH PBS buffer solution and allowed to air dry.
  • a developer TMB (tetramethylbenzidine) substrate liquid matched with the enzyme standard working solution is added to the reaction area of the chip, and 20 ul per well is added for chemiluminescence.
  • the above biomarker proteins of 500 subjects were detected.
  • the results of the test showed that 24 subjects were treated according to the combination category of Table 3 above.
  • the patients were judged to be suspected of pancreatic cancer; the 24 abnormal subjects were further followed up and re-clinical and comprehensive symptom analysis were performed within the next six months.
  • the results showed that 22 of 24 people were diagnosed with pancreatic cancer (of which 6 were of type that had spread, 16 of which were of initial non-proliferation type), and the remaining 2 were not considered to be pancreatic cancer patients, but Other interfering conditions.
  • the detection kit for the above-mentioned markers of the present invention is based on the double-anti-sandwich method, and the detection of the affinity antibody for detecting the above biomarker is carried out, and the detection accuracy has high accuracy and sensitivity. . Moreover, compared with the current CA19-9, CEA, CA242 pancreatic cancer detection markers, it can cover the early stage of pancreatic cancer and not miss the treatment.

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Abstract

一种胰腺癌蛋白生物标记物的检测试剂盒，包括CRP单抗、ICAM-1单抗、OPG单抗和CA19-9单抗，以及用于形成双抗夹心的酶标二抗。一种包括有上述检测试剂盒的胰腺癌检测系统。检测芯片和检测试剂盒以用针对通过"差减法"的方式筛选得到的CRP、ICAM-1、OPG和CA19-9这四种胰腺癌蛋白生物标记物，进行免疫制备特异性CRP单抗、ICAM-1单抗、OPG单抗和CA19-9单抗为基础，并且结合双抗夹心的酶标二抗，通过抗原-抗体的特异性结合并联合双抗夹心的二抗从而对上述生物标记蛋白进行检测，具有比较高的准确性和特异性。

Description

胰腺癌蛋白生物标记物的检测试剂盒及检测系统
技术领域
本发明属于生物检测技术领域，具体涉及一种胰腺癌蛋白生物标记物的检测试剂盒及检测系统。
背景技术
胰腺癌是一种恶性程度很高，诊断和治疗都很困难的消化道恶性肿瘤，约90%为起源于腺管上皮的导管腺癌。通常发病后的患者，5年生存率<1%，是预后最差的恶性肿瘤之一。
导致胰腺癌预后生存率如此低的重要原因在于胰腺癌早期无任何症状，难以在胰腺癌发病潜伏期或者初期被检测发现，从而贻误最佳的治疗时间。主要难以被检测发现是因为在发病初期无任何症状，而且目前所能采用的检测精确度最好的CA19-9、CEA、CA242胰腺癌检测标记物进行检测时，有一定的阳性率，且不具备高特异性。根据统计数据显示，采用上述标记物在胰腺癌孕育期检测（癌变之前）敏感性较低，如发病前1年，有68%的敏感性，发病前两年，敏感性为53%。而采用其他的常规临床技术检测，如B超、CT、MRI、ERCP、PTCD、血管造影、腹腔镜等检查到发现癌变时已处于癌症中晚期，极大耽误了诊断和治疗。
发明内容
本发明实施的目的在于克服现有技术的上述不足，提供一种能够准确性和特异性更好的胰腺癌蛋白生物标记物的检测试剂盒。
为了实现上述发明目的，本发明实施例的技术方案如下：
一种胰腺癌蛋白生物标记物的检测试剂盒，包括CRP单抗、ICAM-1单抗、OPG单抗和CA19-9单抗，以及用于形成双抗夹心的酶标二抗。
在上述双抗夹心的试剂盒的基础上，本发明进一步还提出一种包括上述胰腺癌蛋白生物标记物的检测试剂盒的检测系统。
本发明的检测试剂盒和检测系统，以针对本发明通过“差减法”的方式筛选得到的CRP、ICAM-1、OPG和CA19-9这四种胰腺癌蛋白生物标记物，进行免疫制备特异性CRP单抗、ICAM-1单抗、OPG单抗和CA19-9单抗为基础，并且结合双抗夹心的酶标二抗，通过抗原-抗体的特异性结合并联合双抗夹心的二抗从而对上述生物标记蛋白进行检测，具有比较高的准确性和特异性。
附图说明
下面将结合附图及实施例对本发明作进一步说明，附图中：
图1为本发明实施例检测芯片中固相基质上的点样孔的排布示意图；
图2为本发明实施例胰腺癌蛋白生物标记物的检测芯片封装之后的整体形状示意图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
本发明提出一种胰腺癌蛋白生物标记物，包括CRP(C反应蛋白)、ICAM-1（细胞间黏附分子-1）、OPG（骨保护素）和CA19-9（糖链抗原19-9）。
本发明根据对胰腺癌的检测和检查，提出上述生物标记物，相比现有临床分析的手段和通常用的CA19-9、CEA、CA242胰腺癌检测标记物，本发明的标记物更加准确。本发明的上述生物标记物组合的发现和提出基于本身生物标记物是反映疾病存在和状态的一种可测量的指示物，在疾病、应激和康复的过程中，任何的生理变化最终都能在蛋白水平上得到体现，是疾病研究最直观的手段；并且多个蛋白生物标志物组群与慢性病患者的临床及预后密切相关，表明同步联合测定多个蛋白因子有助于更全面的了解疾病进程。
上述提出的生物标记物组合中的蛋白，属于疾病所特有的调节性蛋白。由于组织液，如血浆中蛋白种类过千，浓度范围跨度高达1010，用最先进性的仪器也只能测到浓度范围跨度103；为攻克健康背景蛋白对疾病特有的调剂蛋白的干扰。在本发明采用差异分析的方法筛选到以上标志蛋白，并进行了验证。具体筛选过程如下：
（1）选取已知身体状况的受试者共210例，其中健康对照55例，胰腺癌初期患者（未转移）65例，发生转移胰腺癌患者90例。（本研究已被伦理审核机构批准，所有受试者均签署知情同意书。）
分别取受试者血液样本2ml，加入1%肝素，于4度，3500RPM离心15min后将血浆分装分别根据患病与否分为健康对照血浆样本、胰腺癌初期（未转移）患者血浆样本和发生转移胰腺癌患者血浆样本；
（2）然后用健康对照血浆样本作为抗原免疫家兔，制备多克隆抗体；并用该制备的多克隆抗体作为亲和介质制备亲和层析柱；
（3）将胰腺癌初期患者样本，发生转移胰腺癌患者样本分别用上述亲和层析柱上样过柱子，收集流穿液。
由于体液如血浆中蛋白种类过千，浓度范围跨度高达1010，目前最先进性的仪器也只能测到浓度范围跨度103；目前用抗体亲和去除样品中20到100多个高丰度蛋白，尽管提高了对低丰度蛋白的检出率，当相对于血浆中1000多个蛋白来说，残留的相对高丰度蛋白对目前癌症所用标志物的ng浓度和癌症发生发展孕育期相关标志物的pg浓度的分析信号仍然存在严重的淹没效果。基于以上原因，本发明采用差减法技术，用健康对照的血浆样本作为抗原免疫家兔，制备抗健康对照血浆蛋白的多克隆抗体，采用这一抗体作为亲和吸附介质制备层析柱，便能将患者血浆样本中的大量健康相关蛋白特异性吸附去除，从而尽可能多地获得疾病相关，排除健康相关高丰度蛋白的干扰。
低丰度差异表达蛋白在富集之后，由于去除了大量的高丰度蛋白的背景干扰，而且在量进行了富集之后，便能用SDS-PAGE和质谱的手段分析和检测出来了。
（4）将上述收集到的流穿液用质谱技术鉴定及定量蛋白
前期筛选阶段，健康对照组、胰腺癌初期患者（未转移）、发生转移胰腺癌患者分别进行质谱鉴定，重复三次，每组蛋白三次重复中共同鉴定到的蛋白分别鉴定到210、320和378个蛋白。
因本发明旨在寻找胰腺癌早期生物标志物，其中仅在胰腺癌初期患者（未转移）、发生转移胰腺癌患者中均出现，但在健康对照中未出现的蛋白有45个。在健康对照组、胰腺癌初期患者（未转移）、发生转移胰腺癌患者均出现的蛋白有179个。本试验主要针对仅在胰腺癌初期患者（未转移）、发生转移胰腺癌患者中共同出现的45个蛋白进行分析。
通过文献报道和生物信息学分析（Gene Ontology和KEGG Pathway分析）对在建康对照中未出现，但在胰腺癌患者血浆中出现的45种进行分析，功能注释。发现CRP(C反应蛋白)，ICAM-1（细胞间黏附分子-1）、OPG（骨保护素）、CA19-9在胰腺癌发生、发展过程中发挥着非常重要的作用。
（5）ELISA法验证
利用ELISA确认上述差异蛋白在正常对照和各疾病组血液中的表达量。具体地说，利用稀释缓冲液将血浆1：100稀释，然后利用针对不同蛋白生物标志物的ELISA板测量不同疾病组血液中生物标志物的含量，每个样本重复测量三次。ELISA技术检测的结果数据用SPSS 14.0软件处理，以平均值±标准差表示。用ANOVA进行正态分布数据的组间比较；用受试者工作特性曲线（ROC）分析各种生物标志物预测的准确性、特异性及灵敏性。检测的结果如下表1，从表1的结果显示，胰腺癌组血浆中CRP(C反应蛋白)、ICAM-1（细胞间黏附分子-1）、OPG（骨保护素）、CA19-9的表达量显著高于健康对照，且随着患者病情的严重（蛋白尿增加）而增加。
表1 ELISA验证4种胰腺癌早期标志物
N值 健康对照(n=55) 胰腺癌初期（无转移）(n=65) 胰腺癌晚期（已转移）(n=90) P值
性别（男/女） 35/20 18/22 29/24 ND
年龄 58±11 60±12 62±5 ND
CRP 16.45mg/ml 67.87mg/ml* 120.2mg/ml* <0.001
ICAM-1 177.1ng/ml 535.4ng/ml* 1010ng/ml* <0.001
OPG 441.7pg/ml 719.5pg/ml* 824.1pg/ml* <0.001
CA19-9 5.42U/ml 36U/ml* 56.04U/ml* <0.001
注：ND: No Difference; *p<0.001 vs. 健康对照；
进一步利用受试者工作特性曲线（ROC）分析胰腺癌患者和健康对照血液中的四种生物标志物，结果表明CRP(C反应蛋白)，ICAM-1（细胞间黏附分子-1）、OPG（骨保护素）、CA19-9曲线下面积（AUC）分别为0.892（p<0.01）、0.858（p<0.01）、0.907（p<0.01）和0.901（p<0.01）。敏感性和特异性分析显示，当选取最高“youden”指数时，血液中四种生物标志物的灵敏度、特异度和浓度截断值的结果见下表2，从表2结果表明这4种蛋白分子可能参与了胰腺癌发生发展过程，为可信的胰腺癌早期生物标记物。
表2 胰腺癌患者和健康对照中各生物标记物的ROC分析对比结果
差异蛋白 灵敏度 特异度 浓度截断值
CRP(C 反应蛋白 ) 80.8% 76.2% 65mg/ml
ICAM-1 71.2% 60.5% 456ng/ml
OPG 79.6% 55.2% 721pg/ml
CA19-9 84.7% 85.8% 39 U/ml
（6）以上述四种蛋白标记物对另100名胰腺癌早期检查验证
对另100名胰腺癌早期患者，通过健康对照的测定结果及四种蛋白分子浓度截断值来确定这4种蛋白标志物相对含量参考正常范围，结合四种生物标志物测量结果来判断。对各种组合进行统计处理，比较不同组合的灵敏度和特异度，其结果参见下表3。其中，表3中符号“∩”表示的组合中，该组合所含的所有生物标志物均为阳性（即高于截断值）时，计算灵敏度和特异度。在符号“∪”表示的组合中，该组合所含的生物标志物有一个为阳性时即判断为阳性，计算灵敏度和特异度。
表3 用于诊断早期胰腺癌蛋白生物标志物组合灵敏度和特异性：
组合 灵敏度 特异度
CA19-9 ∪ OPG 0.933 0.665
CA19-9∩OPG 0.55 0.942
CA19-9 ∪ CRP 0.92 0.594
CA19-9∩CRP 0.547 0.983
CA19-9 ∪ ICAM-1 0.968 0.615
CA19-9∩ICAM-1 0.527 0.941
ICAM-1 ∪ OPG 0.923 0.505
ICAM-1∩OPG 0.541 0.969
ICAM-1 ∪ CRP ∪ OPG 0.989 0.4707
ICAM-1∩CRP∩OPG 0.532 0.983
ICAM-1 ∪ CRP ∪ OPG ∪ CA19-9 0.989 0.475
ICAM-1∩CRP∩OPG∩CA19-9 0.536 0.983
根据上表3的组合检测的结果表明，这表示根据实验目的选择生物标志物的组合，能高效、特异的用于疾病诊断；而且还可以进一步确定，灵敏度高的组合（CA19-9∪OPG；CA19-9∪CRP；CA19-9∪ICAM-1；ICAM-1∪OPG；ICAM-1∪CRP∪OPG；ICAM-1∪CRP∪OPG∪CA19-9）适合于初次筛查；相反，特异度高的组合（CA19-9∩OPG；CA19-9∩CRP；CA19-9∩ICAM-1；ICAM-1∩OPG；ICAM-1∩CRP∩OPG；ICAM-1∩CRP∩OPG∩CA19-9）适合二次检查或三次检查等需要进行可靠性更高的判断检查。例如，若在初次筛查时出现阳性，此时通过特异度较高的组合方法对初次检查结果进行二次判定，最终给出有效且可靠的早期胰腺癌诊断参考结果。
本发明在上述胰腺癌蛋白生物标记物组合的基础上，进一步提出胰腺癌蛋白生物标记物的检测芯片，检测芯片包括：固相载体，以及固定在该固相载体上的CRP单抗，ICAM-1单抗、OPG单抗和CA19-9单抗。
本发明的胰腺癌蛋白生物标记物的检测芯片，通过在固相载体上固定上述蛋白质生物标记物的单克隆抗体，通过对应的单抗捕获能与之特异性结合的待测蛋白，从而实现对存在于血清、血浆、淋巴、间质液、尿液、渗出液、细胞溶解液、分泌液等待测样品中的目的标记蛋白进行分离；之后再经洗涤、纯化、确认和生化分析；即可得到待测样品中的这些生物标记蛋白的差异表达结果。
其中，上述胰腺癌蛋白生物标记物的检测芯片中所包含的4种生物标记蛋白的单抗，在本发明中采用家兔作为免疫体进行免疫制备单克隆抗体，比如以CRP单抗的制备为例，具体包括：
（1）选用4个月左右的雌性小白鼠进行CRP蛋白的免疫注射，注射后该CRP蛋白作为抗原通过血液循环或淋巴循环进入家兔外周免疫器官，刺激相应B淋巴细胞克隆，使其活化、增殖，并分化成为致敏B淋巴细胞；
（2）在免疫达到目标程度时处死家兔，取家兔脾脏；细碎之后酶消化法消化成单细胞悬液，并用肿瘤细胞进行细胞融合杂交；
（3）细胞融合之后用HAT选择性培养基进行选择性培养，筛选成功融合的杂交细胞；
（4）再将成功融合的细胞有限稀释法进行杂交细胞的克隆培养；培养之后采用免疫方法，筛选出能产生CRP蛋白单克隆抗体的阳性杂交瘤细胞；并将该阳性杂交瘤细胞进行体外培养，培养的过程中适时诱导其表达，结束后分离培养液即可得到CRP单抗。
分别制备得到上述CRP单抗，ICAM-1单抗、OPG单抗和CA19-9单抗之后，然后采用共价交联或者非共价交联的方式将其固定至固相载体上即可。
在实施中作为蛋白质芯片的固相载体一般可以从硝酸纤维素膜、尼龙膜、金膜、平板或者玻璃基片中进行选用。在本发明中基于上述单抗所要进行低丰度差异蛋白特异性吸附的目的，优选采用硝酸纤维素膜或玻璃基片。其原因在于，硝酸纤维素膜自身是渗透滤膜，而且经过硝基化的纤维素微孔薄膜，为空结构的容量较大，而且对蛋白质有很强的结合力。
而其中采用玻璃基片主要是方便、廉价、处理简便，而且具有足够的稳定性和惰性，能非常适于本发明产品的大规模商业化生产。但是，在使用中需要针对本发明的特点对该玻璃基片进行处理，因为作为蛋白载体的玻璃基片是玻璃片多采用表面羟基先用N，N-二乙氧基氨丙基三乙氧基硅烷做表面处理；然后偶联抗体蛋白，但是玻璃基片有非常强的非特异性吸附，容易造成很严重的背景干扰，尤其是在本发明中所要检测的上述4种目的蛋白都是低丰度蛋白时，大量的高丰度蛋白的干扰程度会更为强烈。所以本发明采用玻璃基片作为固相载体使用时，用PEG（聚乙二醇）对玻璃基片进行表面处理，用PEG处理的表面比硅化表面信号强度更好，能提高大分子分析元件(如配体)与其结合的能力，并可以减弱非特异性结合；这是因为PEG的空间结构大，从而降低了蛋白质之间的空间位阻，可以提升亲和吸附的特异性。
为了便于本发明上述蛋白芯片检测的对比，本发明的上述蛋白芯片上还固定有阳性对照和阴性对照。其中，阳性对照可以采用IgG单克隆抗体，这个阳性对照的IgG单克隆抗体最好采用上述相同免疫机体制备，比如上述CRP单抗采用家兔为免疫体制备，那么该IgG单克隆抗体也最好对应以家兔为免疫体制备；采用该阳性对照，检测目的蛋白的结合情况。当然，对应上述阳性对照，阴性对照可以采用样品上样的点样稀释液，其不含有任何可结合的蛋白，正好作为空白对照。
为了方便结果对比并防止交叉污染等情况，本发明的上述蛋白芯片的固相载体上设置点样孔，且这些点样孔可以按照矩阵式或者阵列式的方式设置于固相载体的表面上，然后将对应用于检测生物标记蛋白的单抗固定在这些点样孔中。采用该点样孔的结构进行操作检测，一方面点样孔中的蛋白质可以很好地分离，而使交叉污染降到最小；另一方面，孔的凹陷结构可以利于添加和保存点样缓冲液等等，避免点样之后的样品在孵育时出现干化等问题；第三，可以对点样孔的规则和尺寸进行标准化设定，从而利于对点样的量进行横向比对，即可精确控制点样量，还更加利于横向对比及结果的计算。
出于与上述胰腺癌蛋白生物标记物的检测芯片更加便捷的进行情况，本发明还提出一种胰腺癌蛋白生物标记物的检测试剂盒；试剂盒中包括用于特异性检测上述胰腺癌蛋白生物标记物的CRP单抗，ICAM-1单抗、OPG单抗和CA19-9单抗。用与上述蛋白生物标记物对应的单克隆抗体制备试剂盒，进行特异性的检测。用该试剂盒检测可以多种方法实现，比如将上述4种单抗分别作为层析介质制成层析柱，然后对待测样品进行过柱，之后再辅助电泳、质谱等手段进行检测。或者也可以采用酶标抗原的方式进行，将待测的样品蛋白进行酶标之后与上述4种单抗分别进行亲和反应，反应之后对各自单抗结合的酶标抗原进行信号检测。
但是上述两种检测的方法均不方便，因为了更进一步提升检测的便利性和准确性，本发明的上述试剂盒参考双抗夹心法辅助搭配辅助试剂，进一步辅助试剂包括样品稀释缓冲液，本发明中采用含有1.5％～2％BSA（牛血清白蛋白）稳定剂的30~70mM的PBS磷酸盐缓冲液作为该样品稀释缓冲液；采用该样品稀释缓冲液进行样品稀释和洗涤，其中BSA是蛋白的稳定剂，防止蛋白的分解和非特异性吸附；且BSA还能减轻有些不利环境因素如加热，表面张力及化学因素，从而减轻检测过程中有些蛋白的变性。
为了提升试剂盒试剂检测中的结果检测和灵敏性分析，本发明的上述试剂盒进一步按照双抗夹心法的原理还需辅助搭配酶标二抗；在对待测样品点样之后，各目的蛋白会被对应的单抗捕获而亲和吸附结合，此时再添加过量酶标二抗用于与目的蛋白结合，生成夹心体结构；那么结合上的酶标二抗的量就能反应待测样品中目的蛋白的表达情况，通过计算酶标二抗所携带的信号就可得到目的蛋白的结果数据。其中在本发明实施中，上述酶标二抗可以采用比较通用的含HRP（辣根过氧化物酶）的酶标二抗进行，在满足准确性的情形下，也省去了自行设计制造的麻烦。
同时，为了便于对之后酶标二抗信号的检测，在试剂盒中进一步还可以包含有浓缩洗涤液含有1~2% BSA、0.03~0.07%吐温-20的PBS，用于浓缩酶标二抗和信号检测的显色成分。当然，对应需要在之后添加显色剂，TMB（四甲基联苯胺）底物液。
当然，为了便于双夹心法进行检测，在试剂盒中还可以提供固相基质，该固相基质在实施中是类似于上述芯片的基质，充当载体和反应介质的功能。具体的形状和形态也可以参见上述芯片中的描述，在使用的过程中，根据试剂盒说明书的指示将对应的单抗固定至固相基质的点样孔中，然后进行待测样品的检测；当然如果本发明试剂盒中不提供该固相基质载体，实施中也可以借用ELISA试剂盒的点样板进行。
同时，为了便于结果的对比分析，试剂盒中可以提供标准品、阳性对照和阴性对照。对照的设置可以参见上述芯片中的描述，而其用法与待测样样品相同，使用时与待测样品同步进行操作，之后将结果进行比对，提供对比参照。
基于上述胰腺癌蛋白生物标记物的检测试剂盒，本发明还提出一种包括有上述胰腺癌蛋白生物标记物的检测试剂盒的胰腺癌检测系统。本发明的检测系统是基于试剂盒使用时，无法单独直接得到直观的结果，因此进行添加与该试剂盒配合的检测装置设备如CCD检测仪等同类似的这些检测信号设备进行组合；这些装置和仪器可以对试剂盒使用中的试剂的结合变化信号进行检测，最终直接输出明确的直观数据结果，制成一个整体。
本发明的这一包括检测试剂盒的检测系统，将试剂盒产品和与试剂盒能配套使用的设备等等一并组合，通过抗原-抗体的特异性结合从而对上述生物标记蛋白进行检测，最终能完整直接得出检测的直观数据结果，更加便利和直观、准确。
为说明本发明所采用的生物标记物的准确性和应用试剂盒进行检测结果的真实性，以及使上述实施的技术细节和过程方法能更易于本领域技术人员的理解和实施参考，以下通过具体的实施例和实际分析数据进行举例说明。
实施例1
实施例1用于说明采用本发明的上述生物标记物进行胰腺癌检测的准确性和应用试剂盒进行检测结果的真实性。
S11，制备固相基质载体（以硝酸纤维素膜为例进行）：
获取基质硝酸纤维素膜，并按照设计的（点阵形式为5*5阵列，如图1所示，图1中标示1为定位点、2为阳性对照点样孔、7为阴性对照点样孔、3为CRP点样孔、4为ICAM-1点样孔、5为OPG点样孔、6为CA19-9点样孔）的排列方式和点样位置在膜上制备具有检测指标涂层的点样孔，封闭；最后干燥，封装后于4℃保存，之后的形状如图2所示；
S12，在固相基质上固定CRP单抗、ICAM-1单抗、OPG单抗和CA19-9单抗，其中以CRP单抗为例进行说明（其中CRP单抗的制备参见上述实施例中的家兔免疫的制备过程，在此不再赘述；剩余ICAM-1单抗、OPG单抗和CA19-9单抗的固定方式参照该过程进行）：
首先将 0.01~0.5mg/mL的CRP单抗于50mM pH7.2的PBS缓冲液中，制备用于固定的单抗溶液；
用戊二醛活化硝酸纤维素膜载片上的点样孔后，用100μl 先前制备的单抗溶液在点样孔上进行点样，然后盖上盖片，孵育固定5h左右。然后，孵育完成之后用50mM pH7.2的PBS缓冲液洗涤三到四次，去除未结合的抗体和杂质后晾干备用；
为了将戊二醛活化点样孔中要固定CRP单抗的那部分（不与目的蛋白CRP在表面上反应的那部分）封闭，在点样孔上在滴加含有2％BSA的50mM的pH7.2 PBS缓冲液溶液；然后盖上盖片孵育2h；用 BSA 封闭过的载片前后表面用50mM的pH7.2 PBS缓冲液溶液洗涤三到四次，晾干备用。
S13，获取待检测的尿液样品，然后与阴性对照和阳性对照用样品稀释液（含有1.5％～2％BSA稳定剂的30~70mM的PBS缓冲液）稀释3倍后，分别加入检测的点样孔中，每个反应孔加样0.1mL，每个样品做3次平行试验，置37℃孵育1小时使充分反应，后弃去孔内溶液，用浓缩洗涤液洗涤3次；
S14，每孔加入酶标工作液（含HRP的酶标二抗）100ul，37℃孵育30分钟，震荡洗涤3次，每次1分钟；
S15，将与酶标工作液配套的显色剂TMB（四甲基联苯胺）底物液加入芯片反应区域上，每孔加20ul，进行化学发光。
S16，用CCD检测仪进行化学发光的扫描并收集信号，依据扫描结果显示分析检测结果，确定这些生物标记蛋白的表达量是否正常。
采用上述实施的过程，对500名受试者（年龄层次58~70）的上述生物标记蛋白进行检测，检测结果统计发现，其中有24名受试者根据上表3的组合类别的方式进行二次判定为疑似胰腺癌患者；在后续的半年内对这24非正常的受试者进一步进行跟踪和再次临床检验和综合症状分析。结果表明，24人中有22人被确诊为胰腺癌患者（其中，6人属于已经扩散的类型，其中有16人属于初期未扩散的类型），剩余2人认定不属于胰腺癌患者，而是其他干扰病症。
从最终的统计结果看，本发明针对上述标记物的检测试剂盒，其以双抗夹心法为基础，对应构建检测上述生物标记物的亲和抗体进行检测，具有比较高的准确性和敏感性。而且相比目前CA19-9、CEA、CA242胰腺癌检测标记物，能涵盖胰腺癌发病早期的状况而不至于错过治疗。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包括在本发明的保护范围之内。

Claims (13)

  1. 一种胰腺癌蛋白生物标记物的检测试剂盒，其特征在于，包括CRP单抗、ICAM-1单抗、OPG单抗和CA19-9单抗，以及用于形成双抗夹心的酶标二抗。
  2. 如权利要求1所述的胰腺癌蛋白生物标记物的检测试剂盒，其特征在于，所述试剂盒还包括用于固定所述CRP单抗、ICAM-1单抗、OPG单抗和CA19-9单抗的固相基质。
  3. 如权利要求1所述的胰腺癌蛋白生物标记物的检测试剂盒，其特征在于，所述固相基质上具有呈矩阵设置的点样孔，所述CRP单抗、ICAM-1单抗、OPG单抗和CA19-9单抗通过共价交联的方式固定于所述点样孔内。
  4. 如权利要求1所述的胰腺癌蛋白生物标记物的检测试剂盒，其特征在于，所述试剂盒还包括酶标显色剂，且该酶标显色剂为TMB。
  5. 如权利要求1所述的胰腺癌蛋白生物标记物的检测试剂盒，其特征在于，所述试剂盒还包括样品稀释缓冲液，且该样品稀释缓冲液为含有1.5％～2％BSA的30~70mM的PBS缓冲液。
  6. 如权利要求 1所述的胰腺癌蛋白生物标记物的检测试剂盒，其特征在于，所述酶标二抗为HRP的酶标二抗。
  7. 如权利要求6所述的胰腺癌蛋白生物标记物的检测试剂盒，其特征在于，所述试剂盒还包括用于固定所述CRP单抗、ICAM-1单抗、OPG单抗和CA19-9单抗的固相基质。
  8. 如权利要求6所述的胰腺癌蛋白生物标记物的检测试剂盒，其特征在于，所述固相基质上具有呈矩阵设置的点样孔，所述CRP单抗、ICAM-1单抗、OPG单抗和CA19-9单抗通过共价交联的方式固定于所述点样孔内。
  9. 如权利要求6所述的胰腺癌蛋白生物标记物的检测试剂盒，其特征在于，所述试剂盒还包括酶标显色剂，且该酶标显色剂为TMB。
  10. 如权利要求6所述的胰腺癌蛋白生物标记物的检测试剂盒，其特征在于，所述试剂盒还包括样品稀释缓冲液，且该样品稀释缓冲液为含有1.5％～2％BSA的30~70mM的PBS缓冲液。
  11. 如权利要求6所述的胰腺癌蛋白生物标记物的检测试剂盒，其特征在于，所述试剂盒还包括浓缩洗涤液，且该浓缩洗涤液为含有1~2% BSA、0.03~0.07%吐温-20的PBS缓冲液。
  12. 如权利要求6所述的胰腺癌蛋白生物标记物的检测试剂盒，其特征在于，所述试剂盒还包括阳性对照，且该阳性对照为人IgG单克隆抗体。
  13. 一种包括权利要求1所述的胰腺癌蛋白生物标记物的检测试剂盒的胰腺癌检测系统。
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