WO2019157774A1 - 用于npc诊断及预后预测的标志物及其应用 - Google Patents

用于npc诊断及预后预测的标志物及其应用 Download PDF

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WO2019157774A1
WO2019157774A1 PCT/CN2018/083007 CN2018083007W WO2019157774A1 WO 2019157774 A1 WO2019157774 A1 WO 2019157774A1 CN 2018083007 W CN2018083007 W CN 2018083007W WO 2019157774 A1 WO2019157774 A1 WO 2019157774A1
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insl5
nasopharyngeal carcinoma
concentration
ebv
plasma
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PCT/CN2018/083007
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French (fr)
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曾木圣
李世兵
刘颜颜
隋建华
李慧玉
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中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所）
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Priority to US16/766,981 priority Critical patent/US20200319186A1/en
Publication of WO2019157774A1 publication Critical patent/WO2019157774A1/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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/38—Diluting, dispersing or mixing samples
    • 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
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B5/00—ICT specially adapted for modelling or simulations in systems biology, e.g. gene-regulatory networks, protein interaction networks or metabolic networks
    • G16B5/20—Probabilistic models
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/62—Insulins

Definitions

  • the invention relates to the field of medical diagnosis, in particular to a marker for NPC diagnosis and prognosis prediction and an application thereof.
  • Nasopharyngeal carcinoma is one of the most common malignant tumors in China. It is more common in Guangdong, Guangxi, Hunan, Fujian, Jiangxi and other provinces in southern China. The male incidence rate is 2 to 3 times that of females. 3 to 90 years old, but 30 to 50 years old is a high-incidence age zone.
  • EBV-related serum markers are used as markers for the diagnosis of nasopharyngeal carcinoma in high-risk areas of nasopharyngeal carcinoma.
  • EBV shell antigen (VCA) and EA) in serum by immunofluorescence (IFA) was not detected in most normal human serum. Therefore, this technology immediately became the gold standard for nasopharyngeal cancer screening.
  • the traditional immunofluorescence method is subjectively influenced, time-consuming, and highly demanding for the tester, and the results vary with the observer, making it difficult to perform quality control.
  • the method is less sensitive, especially for patients with nasopharyngeal carcinoma who are EBV-negative.
  • ELISA enzyme-linked immunosorbent assay
  • VCA-IgA EB virus shell antigen VCA is used for population screening and early diagnosis in high-risk areas of nasopharyngeal carcinoma, which has high specificity and sensitivity.
  • VCA-IgA has been used so far.
  • the IFA or VCA-IgA ELISA is still used as a first-line auxiliary diagnostic indicator.
  • nasopharyngeal carcinoma The early prevention and treatment of nasopharyngeal carcinoma is the key to improve survival rate.
  • the positive rate of VCA-IgA is 70-95%, and in the normal population, 10% of people are positive for VCA-IgA, meaning 5-30% of patients with nasopharyngeal carcinoma cannot be diagnosed, and 10% cannot rule out the diagnosis. Therefore, it is necessary to develop and research a new diagnostic marker for nasopharyngeal carcinoma, especially for the diagnosis of patients with negative VCA-IgA.
  • the five-year survival rate of patients with nasopharyngeal carcinoma has reached more than 90%, but there is still a lack of indicators for monitoring the prognosis of patients with nasopharyngeal carcinoma, so the development of new types of nasopharyngeal carcinoma
  • the prognostic indicators of patients' survival prognosis can help patients with nasopharyngeal carcinoma to optimize treatment options and further improve survival rate.
  • INSL5 Insulin-like factor 5 Insulin-like peptide 5
  • INS insulin-like factor 5 Insulin-like peptide 5
  • EST expressed sequence tag
  • INSL5 conforms to the structure of the insulin superfamily, including a signal peptide, A chain, B chain and a C-peptide that acts as a linker. After excision of the linked C-peptide chain, it forms a biologically active hormone, A chain and B chain. It is connected by two disulfide bonds, and there is also a disulfide bond in the A chain.
  • INSL5 binds to and activates the receptor by its B chain.
  • Leucine 31 and arginine 35 are the parts that bind to the receptor, but the function of the B chain to bind and activate the receptor must depend on the combination of A and B chains. Spatial structure.
  • the current research on INSL5 suggests that the reduction of INSL5 may cause the sperm motility of male rats to be impaired, the estrous cycle of females is disordered, the number of islet B cells is reduced, leading to impaired glucose tolerance, and studies have shown that INSL5 may be used as a marker for neuroendocrine tumors. The role of INSL5 in NPC has not been reported.
  • the inventors used the ELISA method to detect the concentration of INSL5 in the plasma of the sample, and found that the plasma concentration of INSl5 was significantly different between the two groups of patients with nasopharyngeal carcinoma and healthy people.
  • the ROC curve analysis showed that the area under the INSL5 curve (AUC) was 0.941, the critical value of INSL5 concentration was 2.45 ng/ml, and its sensitivity and specificity were 93.2% and 81.5%, respectively.
  • AUC area under the INSL5 curve
  • the critical value of INSL5 concentration was 2.45 ng/ml
  • its sensitivity and specificity were 93.2% and 81.5%, respectively.
  • the concentration of INSL5 in EBV-positive plasma was significantly higher than that in EBV-negative plasma.
  • the present invention provides a new high-sensitivity and specificity detection index for diagnosis and prognosis prediction of nasopharyngeal carcinoma, especially for EBV-negative patients, for screening and diagnosing nasopharyngeal carcinoma, judging and predicting nasal
  • the prognosis of pharyngeal cancer provides a new way.
  • INSL5 is used as a marker for the diagnosis and prognosis of nasopharyngeal carcinoma.
  • the reagent for quantifying plasma INSL5 is selected from an INSL5-specific ELISA detection reagent.
  • the criteria for determining the high risk of nasopharyngeal carcinoma were determined by plotting the ROC curve, and the criteria for poor prognosis of nasopharyngeal carcinoma were determined by Kaplan-Meier survival analysis.
  • criteria for poor prognosis of nasopharyngeal carcinoma were determined by Kaplan-Meier survival analysis in combination with plasma EBV copy number.
  • a method for predicting or prognosis of nasopharyngeal carcinoma includes the following steps:
  • the criteria for determining the high risk of nasopharyngeal carcinoma are determined by plotting the ROC curve, and the criteria for poor prognosis of nasopharyngeal carcinoma are determined by Kaplan-Meier survival analysis, preferably by Kaplan-Meier survival analysis combined with plasma EBV copy number to determine the nose.
  • Kaplan-Meier survival analysis preferably by Kaplan-Meier survival analysis combined with plasma EBV copy number to determine the nose.
  • the criterion for determining the high risk of nasopharyngeal carcinoma is the Yuden index of the ROC curve.
  • the criterion for determining the high risk of nasopharyngeal carcinoma is: the concentration of INSL5 is higher than 2.45 ng/ml according to the concentration of INSL5;
  • INSL5 concentration is higher than 3.73 ng/ml
  • EBV copy number is higher than 4000 copy/ml.
  • the method for determining the concentration of INSL5 is as follows:
  • Plasma samples are diluted 1:10 with sample dilutions, loaded at 100 ⁇ l/well, and incubated for 2 hours at 37 °C.
  • the detection kit of the invention has the advantages of simple operation, good repeatability, high sensitivity and good specificity, and experimental data shows that the concentration of INSL5 is 2.45 ng/ml, which can effectively distinguish normal human and nasopharyngeal cancer patients.
  • the sensitivity and specificity were 93.2% and 81.5%, respectively.
  • the concentration of INSL5 was 2.25 ng/ml, which can effectively distinguish EBV-negative normal and nasopharyngeal carcinoma patients.
  • the sensitivity and specificity were 97.2% and 91.2%.
  • INSL5 can also be used as a prognostic indicator for nasopharyngeal carcinoma.
  • the cut-off value is 3.73 ng/ml, which is higher than 3.73 ng/ml, indicating a poor prognosis.
  • the five-year survival rate and the non-metastatic survival rate are 81.3% and 88.2%, respectively. It is much lower than the five-year survival rate of 92.2% and 97.3% and the five-year non-metastatic survival rate at low concentrations of INSL5.
  • the concentration of INSL5 combined with EBV DNA copy number (4000 copy/ml is the critical value) can better predict the prognosis of NPC patients.
  • the invention provides a new high sensitivity and specificity detection index for the diagnosis and prognosis prediction of nasopharyngeal carcinoma, and is beneficial to the diagnosis and treatment monitoring of nasopharyngeal carcinoma.
  • Figure 1 is a demonstration that both INSL5 antibodies 46B8 and 23G9 bind to INSL5 and have different epitopes for sandwich ELISA detection of INSL5;
  • Figure 2 shows the concentration of INSL5 in nasopharyngeal carcinoma patients (NPC) and healthy controls EBV positive (Normal) Scatter distribution map of EBV(+)) and healthy control EBV negative (Normal EBV(-)) distribution;
  • Figure 3 shows the results of ROC curve analysis of the concentration of INSL5 in nasopharyngeal carcinoma patients (NPC) and healthy controls (normal);
  • Figure 4 is a ROC curve of INSL5 in the diagnosis of patients with EBV-negative nasopharyngeal carcinoma
  • Figure 5 is a graph of INSL5 alone for survival prognosis analysis
  • Figure 6 is a graph of INSL5 combined with EBV DNA copy number for survival prognosis analysis.
  • mice 6-8 weeks old were immunized with recombinantly expressed mature form of INSL5 fusion protein (with human Fc tag) at a dose of 50 Ug/only/time, boost once every 2 weeks, after 3 immunizations, ELISA detects the antibody titer of mouse serum, mice with higher antibody titer, after a booster immunization, sacrifice the spleen The cell suspension was prepared and subjected to cell fusion with mouse myeloma cell SP2/0.
  • the antigen used in the detection of mouse serum antibody titer by ELISA is the biotinylated mature form of INSL5-His fusion protein. (including His6 label).
  • 46B8 and 23G9 were obtained, showing relatively stronger specific binding activity to the mature form of the INSL5-His fusion protein.
  • 46B8 and 23G9 can also bind to INSL5 (Pre-INSL5), which does not completely excise the intermediate linker precursor form, indicating that their binding epitope is located on the A or B chain of the INSL5 protein.
  • the INSL5 ELISA test kit consists of:
  • ELISA plate ELISA plate pre-coated with INSL5 capture antibody 46B8, prepared as follows:
  • the coating buffer was used to dilute INSL5 antibody 46B8 to 2 ug/ml, and 100 ul of the above diluted 46B8 antibody was added to each well of the ELISA plate, and incubated overnight at room temperature;
  • Coating solution PBS buffer, pH 7.3;
  • Blocking solution 3% BSA, 1 ⁇ PBS;
  • Washing solution 1 ⁇ PBS, 0.05% Tween-20, pH 7.2-7.4;
  • Detection antibody 23G9, biotin labeling, biotin purchased from Thermofisher;
  • Detection reagent HRP-conjugated streptavidin purchased from R&D
  • Substrate developer TMB, purchased from Sigma;
  • Plasma samples were diluted 1:10 with sample dilutions, loaded at 100 ⁇ l/well, and incubated at 37 °C for 2 hours;
  • the INSL5 antibodies 46B8 and 23G9 have different epitopes using competitive ELISA methods and can be used to assemble an ELISA kit to detect INSL5.
  • the inventors recombinantly expressed murine antibodies 46B8-mIgG2a and 23G9-mIgG2a, as well as human murine chimeric antibodies 46B8-hIgG1 and 23G9-hIgG1.
  • the above four recombinantly expressed antibodies still retain good binding ability to the antigen Pre-INSL5 protein.
  • 46B8-hIgG1 could not compete for binding of 23G9-mIgG2a to Pre-INSL5 antigen (Fig. 1C)
  • 23G9-hIgG1 could not compete for binding of 46B8-mIgG2a to Pre-INSL5 antigen (Fig. 1D).
  • 46B8 was used as a capture antibody
  • 23G9 was used as a detection antibody, and different concentrations of INSL5 were detected (Fig. 1E).
  • the ROC curve was plotted by INSL5 concentration values for nasopharyngeal carcinoma patients and normal subjects (Fig. 3).
  • the analysis curve indicates that the concentration of INSL5 is 2.45 ng/ml as the Cut-off value.
  • the sensitivity of INSL5 is 93.2% for the diagnosis of nasopharyngeal carcinoma and the specificity is 81.5%.
  • the area is 0.941.
  • INLB5 can clearly distinguish between nasopharyngeal carcinoma patients and normal subjects in EBV-negative populations, and ROC curves are drawn by the INSL5 concentration of this population (Fig. 4). According to the Yoden index, the maximum cut-off value is set.
  • the analysis curve indicates that the concentration of INSL5 is 2.25 ng/ml.
  • the sensitivity of INSL5 for diagnosis of nasopharyngeal carcinoma is 97.2%, specificity is 91.2%, under the curve.
  • the area is 0.988.
  • the experimental data show that the quantitative analysis of plasma INSL5 concentration provides a new high sensitivity and specificity for the diagnosis of nasopharyngeal carcinoma, especially for EBV-negative patients, which has better diagnostic value and reduces missed diagnosis.
  • INSL5 can be used as Good independent prognostic indicators, but also can be combined with EBV DNA copy number increases prognostic efficacy, providing a new way to diagnose nasopharyngeal carcinoma and predict prognosis of nasopharyngeal carcinoma.

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Abstract

用于NPC诊断及预后预测的标志物及其应用。运用ELISA方法检测样本血浆中INSL5浓度，发现INSl5血浆浓度在鼻咽癌患者和健康人群这两组人群中有显著性统计学差异，ROC曲线分析结果显示，INSL5曲线下面积为0.941，INSL5浓度的临界值为2.45ng/ml，其灵敏度和特异性分别为93.2％、81.5％。在健康人群分组中，EBV阳性血浆中INSL5浓度有明显高于EBV阴性血浆标本。而在所有的EBV阴性的人群中含健康人34例，鼻咽癌患者72例，而INSL5浓度在这两组人群中仍存在显著统计学差异，ROC曲线结果显示，INSL5曲线下面积为0.988，INSL5浓度的临界值为2.25ng/ml，其灵敏度和特异性分别为97.2％和91.2％，能够很好的区分EBV阴性的正常人和鼻咽癌患者。

Description

用于NPC诊断及预后预测的标志物及其应用
技术领域
本发明涉及医学诊断领域，特别涉及用于NPC诊断及预后预测的标志物及其应用。
背景技术
鼻咽癌（NPC）为我国最常见的恶性肿瘤之一，多见于我国南方的广东、广西、湖南、福建、江西等省，男性发病率为女性的2～3倍，我国病例报道年龄分布在3～90岁，但30～50岁是高发年龄区。
由于鼻咽癌患者体内常常含有高滴度抗EB病毒抗体，且其抗体水平随病情发展而变化，因此一些与EBV相关的血清标志物被用作鼻咽癌诊断的标记在鼻咽癌高发地区进行人群筛查诊断。其中发现较早，用途最广的莫过于通过免疫荧光法（IFA）检测血清中EBV壳抗原（VCA）和早期抗原（EA），而大部分正常人血清中未能检出。因此，这项技术随即成为鼻咽癌筛查的金标准。然而传统免疫荧光法受主观影响大、耗时长、对检测者熟练程度要求高，结果随观察者不同而改变，难以进行质量质控。同时该方法敏感性较低，尤其是容易漏诊EBV阴性的鼻咽癌患者。这些局限性被随之孕育而生的酶联免疫吸附测定（ELISA）所突破，作为第二代鼻咽癌血清学检查技术，ELISA具有更高的准确性，便于实现自动化，运用试剂盒快速进行诊断过程，更适于大规模人群筛查。EB病毒壳抗原VCA用于鼻咽癌高发地区人群筛查及早期诊断，其具有较高的特异性及敏感度，至今VCA-IgA IFA或VCA-IgA ELISA仍作为一线的辅助诊断指标。
鼻咽癌的三早防治是提高生存率的关键，然而在鼻咽癌中，VCA-IgA阳性率为70-95%，而在正常人群中亦有10%的人存在VCA-IgA阳性，意味着有5-30%的鼻咽癌患者无法明确诊断，同时有10%的人无法排除诊断。因此开发和研究新型的鼻咽癌诊断标记物是很有必要的，尤其是对VCA-IgA阴性的患者的诊断具有重要的临床价值。此外，由于现阶段放化疗技术的提高，鼻咽癌患者的五年生存率已达90%以上，但是目前仍缺乏对鼻咽癌患者预后状况进行监测的指标，因此开发新型的对鼻咽癌患者生存预后具有预测作用的指标有助于鼻咽癌患者更加优化治疗方案，进一步提高生存率。
INSL5（Insulin-like factor 5胰岛素样肽5）是胰岛素（INS）超家族的成员，1999年首次由Conklin等在研究INS超家族成员B链的半胱氨酸序列时，通过搜索表达序列标签（EST）数据库时发现。INSL5符合胰岛素超家族的结构，包括一个信号肽、A链、B链和起连接作用的C肽，在切除了起连接作用的C肽链后，形成具有生物活性的激素，A链和B链通过2个二硫键连接，同时A链内还有一个二硫键。INSL5结合并激活受体的是其B链，亮氨酸31和精氨酸35是其与受体结合的部分，但B链结合并激活受体的功能必须依赖A链和B链结合形成的空间结构。目前关于INSL5的研究提示INSL5减少会引起雄鼠精子活力受损、雌鼠发情周期紊乱，胰岛B细胞数量减少，导致糖耐量受损等，同时有研究表明INSL5可能作为神经内分泌瘤的标记物。INSL5在NPC中的作用未见报道。
发明内容
本发明的目的在于提供一种用于NPC诊断及预后预测的标志物及其应用。
发明人运用ELISA方法检测样本血浆中INSL5浓度，发现INSl5血浆浓度在鼻咽癌患者和健康人群这两组人群中有显著性统计学差异，ROC曲线分析结果显示，INSL5曲线下面积（AUC）为0.941，INSL5浓度的临界值为2.45ng/ml，其灵敏度和特异性分别为93.2%、81.5%。同时在健康人群分组中，EBV阳性血浆中INSL5浓度有明显高于EBV阴性血浆标本。而在所有的EBV阴性的人群中含健康人34例，鼻咽癌患者72例，而INSL5浓度在这两组人群中仍存在显著统计学差异，ROC曲线结果显示，INSL5曲线下面积为0.988，INSL5浓度的临界值为2.25ng/ml，其灵敏度和特异性分别为97.2%和91.2%，能够很好的区分EBV阴性的正常人和鼻咽癌患者。
在NPC预后预测方面：利用Kaplan-Meier生存分析发现INSL5高浓度和低浓度组之间患者的生存期和无转移生存期均存在显著性差异，INSL5浓度的临界值为3.73ng/ml，其中浓度高于3.73ng/ml的患者五年生存率为81.3%，同时远处转移的发生率为11.8%；而INSL5浓度低于3.73ng/ml的患者五年生存率为92.2%，同时远处转移的发生率为2.7%；此外联合INSL5浓度和血浆EBV DNA拷贝数，发现INSL5浓度高于3.73ng/ml同时EBV拷贝数高于4000copy/ml时能够更好的预测NPC患者生存期和无转移生存期。
因此本发明为鼻咽癌的诊断和预后预测提供了新的高灵敏度与特异性的检测指标，尤其对EBV阴性患者有较好的诊断价值，为筛查和诊断鼻咽癌、判断和预测鼻咽癌预后提供了一个新途径。
本发明所采取的技术方案是：
INSL5作为鼻咽癌诊断及预后预测标志物的应用。
定量血浆INSL5浓度的试剂在制备鼻咽癌诊断及预后预测试剂中的应用。
作为上述应用的进一步改进，定量血浆INSL5的试剂选自INSL5特异的ELISA检测试剂。
作为上述应用的进一步改进，通过绘制ROC曲线确定鼻咽癌高风险的判定标准，通过Kaplan-Meier生存分析确定鼻咽癌不良预后的标准。
作为上述应用的进一步改进，通过Kaplan-Meier生存分析联合血浆EBV拷贝数确定鼻咽癌不良预后的标准。
一种鼻咽癌诊断或预后预测的方法，包括如下步骤：
1) 确定待测血浆样品中INSL5的浓度；
2) 根据测得的INSL5的浓度，判定待测样品是否为鼻咽癌高风险样品和/或预测其预后情况。
作为上述方法的进一步改进，通过绘制ROC曲线确定鼻咽癌高风险的判定标准，通过Kaplan-Meier生存分析确定鼻咽癌不良预后的标准，优选通过Kaplan-Meier生存分析联合血浆EBV拷贝数确定鼻咽癌不良预后的标准。
作为上述方法的进一步改进，鼻咽癌高风险的判定标准为ROC曲线的Yuden指数。
作为上述方法的进一步改进，鼻咽癌高风险的判定标准为：以INSL5浓度计，INSL5浓度高于2.45ng/ml；
鼻咽癌不良预后的标准为：以INSL5浓度计，INSL5浓度高于3.73ng/ml，或INSL5浓度高于3.73ng/ml且EBV拷贝数高于4000copy/ml。
作为上述方法的进一步改进，INSL5浓度的测定方法如下：
1) 血浆样本用样本稀释液以1:10稀释，以100μl/孔加样，37℃中孵育2小时
2) PBST洗板3次，最后一次控干，加入稀释好的biotin标记的检测抗体23G9，100μl/孔，37℃中孵育2小时；
3) PBST洗板3次，最后一次控干，加入稀释好的偶联HRP的链霉亲和素，100μl/孔，常温避光孵育20min；
4) PBST洗板5次，最后一次控干，加入显色底物TMB，100μl/孔，37℃显色不超过20min；
5) 加入50μl 2M H2SO4 终止反应；
6) 终止反应后10min内酶标仪上读取450nm 波长下的OD值，并根据标准曲线计算出待测样品中INSL5浓度。
本发明的有益效果是：
本发明的检测试剂盒，操作简单，重复性好，具有灵敏度高，特异性好的优点，实验数据表明，INSL5浓度以2.45ng/ml为临界值，可有有效区分正常人和鼻咽癌患者，其灵敏度和特异性分别为93.2%、81.5%；同时INSL5浓度以2.25ng/ml为临界值，可以有效区分EBV阴性的正常人和鼻咽癌患者，其灵敏度和特异性分别为97.2%和91.2%。
此外INSL5还可以作为鼻咽癌预后预测的指标，其临界值为3.73ng/ml，高于3.73ng/ml预示着预后不良，五年生存率和无转移生存率分别为81.3%、88.2%，远低于INSL5低浓度时92.2%和97.3%的五年生存率和五年无转移生存率。同时INSL5浓度联合EBV DNA拷贝数（4000copy/ml为临界值）能够更好的对NPC患者进行预后预测。
本发明为鼻咽癌的诊断和预后预测提供新的高灵敏度与特异性的检测指标，有利于鼻咽癌的诊断和治疗监测。
附图说明
图1是证实INSL5抗体46B8和23G9都能和INSL5结合，且具有不同的表位，以用于夹心ELISA检测INSL5；
图2是INSL5浓度在鼻咽癌患者（NPC）、健康对照组EBV阳性(Normal EBV(+))和健康对照组EBV阴性（Normal EBV（-））分布的散点分布图；
图3是鼻咽癌患者（NPC）与健康对照组（normal）检测INSL5浓度，进行ROC曲线分析的结果；
图4是INSL5诊断EBV阴性鼻咽癌患者的ROC曲线；
图5是INSL5单独用于生存预后分析的曲线；
图6是INSL5联合EBV DNA拷贝数用于生存预后分析的曲线。
具体实施方式
INSL5抗体的制备：
利用重组表达的成熟形式的INSL5融合蛋白（带有人Fc标签）常规腹腔注射免疫6～8周龄的BALB/c雌性小鼠，剂量为50 ug/只/次，每隔2周加强免疫一次，经过3次免疫后，ELISA检测小鼠血清的抗体滴度，含有较高抗体滴度的小鼠，在经过一次加强免疫后，处死取脾脏，制成细胞悬液与小鼠骨髓瘤细胞SP2/0进行细胞融合。ELISA检测小鼠血清抗体滴度时使用的抗原为生物素化的成熟形式的INSL5-His融合蛋白 (含His6标签)。细胞融合后进行大量ELISA筛选和亚克隆化，最终获得46B8和23G9两株克隆，表现出对成熟形式的INSL5-His融合蛋白相对更强的特异性结合活性。另外，46B8和23G9它们也可以跟没有完全切除中间连接肽前体形式的INSL5（Pre-INSL5）结合，说明它们的结合表位位于INSL5蛋白的A链或B链上。
INSL5 ELISA检测试剂盒组成：
ELISA板：预包被INSL5捕获抗体46B8的ELISA板，其制备方法如下：
1) 包被缓冲液将INSL5抗体46B8稀释成2ug/ml，在ELISA板的每孔加入100ul上述稀释的46B8抗体，常温孵育过夜；
2) 甩干包被液，PBST洗涤3次，最后一次控干；加入3%BSA封闭液，每孔300ul，常温封闭2h；
3) PBST洗板3次，最后一次拍干，放于4℃保存备用。
包被液：PBS缓冲液，pH 7.3；
封闭液：3%BSA，1×PBS；
稀释液：1×PBS，pH 7.2-7.4；
洗涤液：1×PBS，0.05%吐温-20，pH7.2-7.4；
标准品：INSL5粉末溶解后，稀释至不同梯度的浓度，作为标准品，购买自康肽生物；
检测抗体：23G9，进行生物素标记，生物素购买自Thermofisher；
检测试剂：HRP偶联的链霉亲和素，购买自R&D公司
底物显色剂：TMB，购买自Sigma；
终止液：2M H2SO4 。
鼻咽癌患者
收集中山大学肿瘤防治中心2009年1月～2012年12月期间住院及门诊鼻咽癌患者339例。入组标准：病理诊断已确诊II/III型鼻咽癌（基于1978年世界卫生组织分类系统）；有准确的临床分期（基于美国AJCC癌症分期手册第七版）；样本均来自南方鼻咽癌高发地区的病人（广东、广西、江西、湖南、福建、四川）；样本血浆为治疗前抽取；患者具有完整人口统计学资料，病历资料，包括血清VCA-IgA免疫酶法及血浆EBV DNA定量检测结果。其中EBV阴性者75例，EBV阳性者209例。
健康人群
随机抽取中山大学职工体检血浆样本65份，排除其他疾病，保留所有健康人群资料，包括VCA-IgA免疫酶法检测结果。其中EBV阴性者34名，EBV阳性者31名。
检测方法：
1) 血浆样本用样本稀释液以1:10稀释，以100μl/孔加样，37℃中孵育2小时；；
2) PBST洗板3次，最后一次控干，加入稀释好的biotin标记的检测抗体23G9，100μl/孔，37℃中孵育2小时；
3) PBST洗板3次，最后一次控干，加入稀释好的偶联HRP的链霉亲和素，100μl/孔，常温避光孵育20min；
4) PBST洗板5次，最后一次控干，加入显色底物TMB，100μl/孔，37℃显色不超过20min；
5) 加入50μl 2M H2SO4 终止反应；
6) 终止反应后10min内酶标仪上读取450nm 波长下的OD值，并根据标准曲线计算出待测样品中INSL5浓度。
检测结果及分析：
1．抗体验证：利用竞争性ELISA方法证实INSL5抗体46B8和23G9具有不同表位，可用于组装ELISA试剂盒检测INSL5。
发明人重组表达了鼠抗体46B8-mIgG2a和23G9-mIgG2a，以及人鼠嵌合抗体46B8-hIgG1和23G9-hIgG1。如图1A和1B所示，上述四种重组表达抗体仍然保持了和抗原Pre-INSL5蛋白良好的结合能力。然后利用竞争性ELISA方法，发现46B8-hIgG1不能竞争23G9-mIgG2a与Pre-INSL5抗原的结合（图1C），同时23G9-hIgG1亦不能竞争46B8-mIgG2a与Pre-INSL5抗原的结合（图1D）。进一步以46B8作为捕获抗体，23G9作为检测抗体，可以检测不同浓度的INSL5（图1E）。
2．血浆标本检测：
选择鼻咽癌病人339例，健康对照65例进行血浆样本检测（血浆来源于中山大学肿瘤防治中心），根据标准品浓度计算出INSL5浓度后，用SPSS(20.0)统计学软件进行分析。经统计，该数据为非正态分布，因此用Wilcoxon秩和检验。
Wilcoxon秩和检验结果显示INSL5血浆浓度在两组人群中有显著性统计学差异，鼻咽癌患者的血浆INSL5浓度明显高于正常人组（p值小于0.0001），同时正常人组中EBV阳性组INSL5浓度显著高于EBV阴性组，其散点分布图见附图2。
表 1 、 INSL5 在鼻咽癌及正常组中的比较
分组 中位浓度（ ng/ml ） 四分位间距 P
<0.001
鼻咽癌 4.1464 1.886
正常 EBV （ + ）组 2.2854 0.7343
正常 EBV （-）组 1.6524 0.3292
通过对鼻咽癌患者及正常人组的INSL5浓度值绘制ROC曲线（附图3）。
根据约登指数(Yuden index)最大时设定Cut-off值，分析曲线表明选择INSL5浓度为2.45ng/ml作为Cut-off值，INSL5浓度用于诊断鼻咽癌的敏感度93.2%，特异性为81.5%，曲线下面积为0.941。
此外在EBV阴性的人群中，INSL5也能明显区分鼻咽癌患者和正常人，通过对这一类人群的INSL5浓度绘制ROC曲线（附图4）。根据约登指数最大是设定cut-off值，分析曲线表明此时选择INSL5的浓度为2.25ng/ml，测试INSL5用于诊断鼻咽癌的敏感度为97.2%，特异性91.2%，曲线下面积为0.988。
在对拥有预后信息的304例病例进行分析发现INSL5是独立的预后因素，以3.73ng/ ml作为临界值，较高浓度预示着预后不良，五年生存率和无转移生存率分别为81.3%、88.2%，远低于INSL5低浓度时92.2%和97.3%的五年生存率和无转移生存率（附图5）。
同时INSL5浓度联合EBV DNA拷贝数（EBV DNA拷贝数以4000copy/ml为临界值）能够更好的对NPC患者进行预后预测（附图6）。
综上，实验数据表明，血浆INSL5浓度的定量分析为鼻咽癌的诊断提供新的高灵敏度与特异性的检测指标，尤其对EBV阴性患者有较好的诊断价值，减少漏诊，同时INSL5可以作为很好的独立的预后预测指标，同时也可以联合EBV DNA拷贝数增加预后预测效能，为诊断鼻咽癌和预测鼻咽癌预后信息提供了一个新途径。

Claims (10)

  1. INSL5作为鼻咽癌诊断及预后预测标志物的应用。
  2. 定量血浆INSL5浓度的试剂在制备鼻咽癌诊断及预后预测试剂中的应用。
  3. 根据权利要求2所述的应用，其特征在于：定量血浆INSL5的试剂选自INSL5特异的ELISA检测试剂。
  4. 根据权利要求2所述的应用，其特征在于：通过绘制ROC曲线确定鼻咽癌高风险的判定标准，通过Kaplan-Meier生存分析确定鼻咽癌不良预后的标准。
  5. 根据权利要求4所述的应用，其特征在于：通过Kaplan-Meier生存分析联合血浆EBV拷贝数确定鼻咽癌不良预后的标准。
  6. 一种鼻咽癌诊断或预后预测的方法，包括如下步骤：
    1) 确定待测血浆样品中INSL5的浓度；
    2) 根据测得的INSL5的浓度，判定待测样品是否为鼻咽癌高风险样品和/或预测其预后情况。
  7. 根据权利要求6所述的方法，其特征在于：通过绘制ROC曲线确定鼻咽癌高风险的判定标准，通过Kaplan-Meier生存分析确定鼻咽癌不良预后的标准，优选通过Kaplan-Meier生存分析联合血浆EBV拷贝数确定鼻咽癌不良预后的标准。
  8. 根据权利要求6所述的方法，其特征在于：鼻咽癌高风险的判定标准为ROC曲线的Yuden指数。
  9. 根据权利要求6所述的方法，其特征在于：
    鼻咽癌高风险的判定标准为：以INSL5浓度计，INSL5浓度高于2.45ng/ml；
    鼻咽癌不良预后的标准为：以INSL5浓度计，INSL5浓度高于3.73ng/ml，或INSL5浓度高于3.73ng/ml且EBV拷贝数高于4000copy/ml。
  10. 根据权利要求 6 ～ 9 任一项所述的方法，其特征在于： INSL5 浓度的测定方法如下：
    1) 血浆样本用样本稀释液以 1:10 稀释，以 100μl/ 孔加样， 37 ℃中孵育 2 小时
    2) PBST 洗板 3 次，最后一次控干，加入稀释好的 biotin 标记的检测抗体 23G9 ， 100μl/ 孔， 37 ℃中孵育 2 小时；
    3) PBST 洗板 3 次，最后一次控干，加入稀释好的偶联 HRP 的链霉亲和素， 100μl/ 孔，常温避光孵育 20min ；
    4) PBST 洗板 5 次，最后一次控干，加入显色底物 TMB ， 100μl/ 孔， 37 ℃显色不超过 20min ；
    5) 加入 50μl 2M H2SO4 终止反应；
    6) 终止反应后 10min 内酶标仪上读取 450nm 波长下的 OD 值，并根据标准曲线计算出待测样品中 INSL5 浓度。
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CN114121142B (zh) * 2021-09-02 2023-10-31 四川大学华西医院 一种新型基因修饰增强型ny-eso-1特异型tcr-t模型构建方法及应用

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