APE1血清学检测方法的建立及其在肺癌诊断中的应用
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摘要
肺癌的发病率和死亡率在我国一直位居首位,并呈逐年增多的趋势,若能早期诊断并及时治疗,90～95%的肺癌是可以治愈的,因此肺癌的早期诊断是提高肺癌治愈率最有效的方法。肿瘤标志物作为肿瘤早期诊断的一种无创性检测,在肿瘤普查、诊断、判断预后、评价疗效和高危人群随访观察等方面都具有较大的实用价值。近年来,随着生物技术(包括蛋白质组、ELISA、RT-PCR、FISH和SELDI-TOF等)的飞速发展,发现了许多有应用前景的肿瘤标志物,但目前无任何一种标志物可准确诊断肺癌和预测预后,多肿瘤标志物联合诊断是提高肺癌诊断准确率的可行策略之一。寻找特异度和灵敏度高的肿瘤标志物联合检测成为目前的研究热点。
脱嘌呤/脱嘧啶核酸内切酶(APE1)是碱基切除修复(BER)途径中的一个限速酶,具有DNA损伤修复和氧化还原等多种功能,参与多种与细胞重要功能有关的反应,其功能异常可能导致肿瘤形成。研究表明,APE1基因表达具有器官和组织特异性,在肺癌组织中APE1表达水平较正常组织显著升高,且有明显的易位表达。APE1表达定位的改变及表达强度的变化可能是细胞表型异常的决定因素。由于肿瘤细胞增殖过快,不断有细胞发生凋亡和坏死,使细胞内大量蛋白质释放入血,血清中APE1蛋白含量可能升高。理论上,APE1表达水平和定位的改变会刺激机体产生抗APE1的自身抗体。目前认为血清抗体的产生是机体对肿瘤产生的一种特异性的免疫反应,间接反映了肿瘤的发展和演变。对APE1进行血清学检测可能有助于肿瘤的临床诊断,目前对APE1的检测大都为免疫组化或免疫印迹,其定位研究较为透彻,血清学检测在国内外尚未见报道。本研究首先制备兔抗人APE1多克隆抗体,然后建立改良的双抗体夹心ELISA和间接ELISA,检测血清APE1蛋白和抗体的表达,并探讨其与多肿瘤标志物联合检测在肺癌中的诊断作用及其临床应用价值。
研究目的
1.制备兔抗人APE1多克隆抗体,特异性亲和纯化并对其生物学特性进行鉴定;
2.建立双抗体夹心ELISA检测血清中APE1蛋白并结合蛋白芯片检测结果探讨其在肺癌诊断中的作用;
3.建立间接ELISA检测血清中APE1抗体并结合蛋白芯片检测结果探讨其临床应用价值。
研究内容和方法
1.兔抗人APE1多克隆抗血清的制备
用纯化的全长APE1蛋白免疫新西兰大白兔,采用一种快速免疫法制备APE1多克隆抗体,ELISA追踪检测抗体效价,Protein A亲和层析法纯化特异性IgG抗体,Western blot和免疫组化检测抗体的特性,为进一步建立双抗体夹心ELISA提供抗体。
2.改良的双抗体夹心ELISA检测血清APE1蛋白方法的建立
以鼠抗人APE1单克隆抗体包被高亲和力酶标板,APE1全长蛋白为标准品,兔抗人APE1多克隆抗体为检测抗体,HRP标记羊抗兔IgG为酶标抗体建立双抗体夹心ELISA,棋盘滴定法确定各抗体的最佳工作浓度并对ELISA方法的灵敏度、特异度和稳定性进行检测。采用所建立的ELISA方法检测肺癌患者和健康体检者血清并结合蛋白芯片检测结果进行统计学分析,探讨其临床意义。
3.间接ELISA检测血清APE1抗体方法的建立
以全长APE1蛋白包被高结合力酶标板,HRP标记羊抗人IgG为酶标抗体建立间接ELISA。采用所建立的ELISA方法检测肺癌患者和健康体检者血清并结合蛋白芯片检测结果进行统计学分析,从不同角度探讨其临床应用价值。
研究结果
1.成功制备了兔抗人APE1多克隆抗体。ELISA检测显示该抗体的效价达到1:128000,相对亲和力常数为8.96×10-6mol/L。Western blot和免疫组化显示该抗体能与APE1蛋白特异性结合。此外,该抗体还可用于小鼠和大鼠组织APE1蛋白的检测。
2.建立的夹心ELISA检测APE1蛋白的线性范围为8.0～200 ng/mL;批内变异系数为8.37%～8.97%,批间变异系数为10.47%～14.57%;平均回收率为90.83%。血清中APE1蛋白含量呈偏态分布,健康体检者和肺癌患者血清APE1含量95%可信区间分别为3.67ng/mL～16.58ng/mL和7.06ng/mL～24.90ng/mL,肺癌患者血清APE1蛋白水平明显高于健康体检者(P<0.05)。
3.回顾性分析发现,CEA、CA125和CA242三项标志物对肺癌诊断的灵敏度和准确率较高。APE1与这三项指标联合检测对肺癌诊断的灵敏度为68.57%,特异度为80.00%,准确率为74.15%。联合APE1检测较单一标志物检测的诊断灵敏度和准确率都有所提高,但是同时也降低了诊断的特异度。
4.建立的间接ELISA在血清稀释度为1:3200时仍能检测的到。批内变异系数平均为8.52%,批间变异系数平均为9.09%。肺癌患者血清APE1抗体水平明显高于健康体检者(P<0.05),APE1抗体阳性率分别为34.49%和3.43%,二者有显著性差异(P<0.05);但APE1抗体在不同性别,不同吸烟状态,不同TNM分期和不同组织病理学分型上并无显著性差异(P>0.05)。
5. APE1抗体还与治疗疗效紧密相关,化疗后APE1抗体水平显著高于治疗前;治疗有应答者(CR+PR)血清APE1抗体在化疗后比化疗前显著升高(P<0.05),而无应答者(SD+PD)血清APE1抗体水平在化疗前后没有显著性差别(P>0.05),提示血清APE1抗体可能对预测肺癌的化疗敏感性有重要参考价值。
6.血清APE1抗体与肺癌血清检测常用的肿瘤标志物CEA、CA125、和CA242联合检测的诊断灵敏度、特异度和准确率分别为71.17%、95.57%和84.03%。
结论
1.成功制备了兔抗人APE1多克隆抗体并进行了特异性亲和纯化,抗体效价和纯度均很高;Western blot和免疫组化显示此抗体特异性良好,可满足实验要求。
2.首次成功建立了改良的双抗体夹心ELISA检测APE1蛋白,所建立的ELISA方法的灵敏度、特异度、精确度、批内批间差异和稳定性均满足实验要求。
3.健康体检者和肺癌患者血清中均能检测到APE1蛋白,但表达量不同,肺癌患者血清APE1蛋白明显高于健康体检者;APE1与CEA、CA125、和CA242联合检测能提高诊断的准确率,提示APE1有望成为一种新的肿瘤标志物。
4.首次成功建立了间接ELISA检测血清APE1抗体,肺癌患者血清APE1抗体水平明显高于健康体检者,但APE1抗体在不同性别、吸烟状态、TNM分期和组织病理学分型上并无显著性差异。
5.血清APE1抗体还与治疗疗效紧密相关,血清APE1抗体可能对预测肺癌的化疗敏感性有重要参考价值。
6.血清APE1抗体与肺癌血清检测常用的肿瘤标志物CEA、CA125、和CA242联合检测可提高诊断的灵敏度和准确率。APE1抗体有可能成为肺癌辅助诊断的指标之一。
Lung cancer is the most common malignant tumor with high morbidity and mortality in China, and the morbidity is increasing year by year. The World Health Organization pointed out, 90~95 percent cancer is curable with early diagnosis and timely treatment. Early diagnosis of lung cancer is still the most effective way to improve the survival rate. Tumor markers have been used in the tumor screen, diagnosis, prognosis, evaluation of efficacy and the follow-up of high-risk groups. With recent advances in biotechnology such as proteomics, FISH, ELISA, RT-PCR, and SELDI-TOF, many promising biomarkers have been identified and are currently under investigation and validation. However, it is unlikely that any tumor marker will perfectly diagnose lung cancer. At present, the recombination of multiple predictive factors will be a realistic way to improve lung cancer in screening and staging. At present, searching for more specific and sensitive tumor markers is the most promising way to improve cancer diagnosis.
APE1 is a multifunctional protein that not only repairs the apurinic/apyrimidinic sites of DNA lesions, acting as a rate-limiting enzyme of BER pathway, but also functions as a reduction-oxidation factor protecting cells against the toxic effects. We believe the dysfunction of APE1 have the potential in increasing susceptibility of cancer. Most studies show that, lung cancer existed APE1 overexpression and altered localization, these alteration are likely to be the abnormal cell phenotype determinants. Since the tumor cell multiplication is excessively quickly, within unceasingly cell apoptosis and necrosis, a large number of cellular proteins released into the blood, so we proposed that APE1 protein in serum may be increased. APE1’s altered expression, primarily at the protein or subcellular localization level may stimulate organism to produce antibodies. As the antibody amount is more than the antigen and antibody exist in serum for a long time, so the detection of specific antibody may allow for identification of an underlying tumor. APE1 is expected to become a new biomarker for lung cancer.
To our knowledge, there is no report about APE1 proteins or antibodies level in serum. Studies on screening APE1 alterations were mostly by immunohistochemical staining or western blot. So far, this is the first examination of serum APE1 level in relation to cancer diagnosis. Our main objective of this prospective study is 1) to prepare rabbit anti-human APE1 polyclonal antibody 2) to evaluate clinical value of serum APE1 protein and antibody or in combination with CEA, CA125 and CA242 in distinguishing between lung cancer patients and healthy.
Objective:
1. To prepare rabbit anti-human APE1 polyclonal antibody and then characterize it.
2. To establish a sandwich ELISA for quantitative measurement of APE1 protein, measure serum APE1 level in healthy and lung cancer patients, and to study its clinical application in combination with CEA, CA125 and CA242.
3. To detect serum APE1 antibody in lung cancer patients and healthy by indirect ELISA, monitor patients before and after chemotherapy, combine with CEA, CA125 and CA242 to evaluate if serum APE1 antibody can be used as a new tumor marker and predictor of therapeutic efficacy.
Materials and Methods:
1. Preparation and characterization of rabbit anti-human APE1 polyclonal antibody Rabbit anti-human APE1 polyclonal antibody was prepared by a modified rapid immune procedure followed by purification of specific avidity column. The efficacy of this polyclonal antibody was certificated by ELISA, Western blot and immunohistochemistry.
2. Establishment and primary application of a sandwich ELISA for quantitative measurement of APE1
The assay was based on the sandwiching of the antigen between a monoclonal mouse anti-APE1 antibody, pre-coated on a 96 well polystyrene plate, and a polyclonal rabbit anti-APE1 antibody, which was then detected with a peroxidase-labeled goat anti-rabbit antibody. The optimal concentrations were defined by titration. Standard curve was performed using purified APE1 protein and was judged by sensitivity, reproducibility and recovery rate. The sandwich ELISA was tried to measure the APE1 levels in healthy and lung cancer patients, and then combined with CEA, CA125 and CA242 to study its clinical application.
3. Establishment and primary application of indirect ELISA in lung cancer and healthy Indirect ELISA was carried out to detect APE1-Abs in 345 lung cancer patients and 350 healthy. Ninety-one patients were monitored before and after chemotherapy. The data was analyzed by independent t-test, ROC curve and chi-square test. APE1 antibody was combined with CEA, CA125 and CA242 to study its clinical application.
Results:
1. The titer of the antiserum obtained in this experiment determined by ELISA was up to 1∶128 000.The kaff value of the antibody was 8.96×10-6mol/L. Western blot results showed the antibody has high specificity to APE1 protein. The final purified antibody was highly specific to native form of APE1 protein in human, mouse and rat.
2. In the range of 8.0 ng/mL to 200 ng/mL, the APE1 antigen showed a good linearity in the standard curve. The sensitivity of this assay was 2.0 ng/mL. The intra-assay precision and inter-assay precision were 8.37%～8.97% and 10.47%～14.57% respectively. The average recovery rate was 90.83 %. APE1 protein levels in serum were skewed distribution, The 95% CI of serum APE1 concentration in healthy and lung cancer patients were 3.67ng/mL～16.58ng/mL and 7.06ng/mL～24.90ng/mL respectively. The results demonstrated a significant increase of serum APE1 level in cancer patients compared to healthy (P<0.05).
3. Retrospective analysis found that, CEA, CA125 and CA242 were more specific for diagnosis of lung cancer and had higher positive rate. APE1 combined with these three makers can elevate the diagnostic sensitivity and correct rate. In this study, the diagnostic sensitivity, specificity and correct rate were 68.57%, 80.00% and 74.15%, respectively. The diagnostic sensitivity and accuracy were improved than a single indicator, but at the same time the specificity of diagnosis were reduced.
4. Serum APE1-Abs level of lung cancer patients was significantly higher than that of healthy volunteers (P=0.000). For each possible cut-point, the resulting sensitivity and specificity were indicated as a point on the ROC graph. The area under ROC was 0.735. One hundred and nineteen (34.49%) lung cancer patients and 12 (3.43%) healthy were positive with APE1-Abs. But variables such as sex, smoking status, TNM stages and histopathological types were not associated with APE1-Abs (P>0.05, respectively).
5. We also found serum APE1-Abs were significantly associated with response to therapy. Serum APE1-Abs of responders were significantly higher after chemotherapy (P=0.000), while APE1-Abs level in non-responders, the difference was not significant (P=0.393).
6. Diagnostic sensitivity, specificity and correct rate of APE1 antibody with combination of CEA, CA125 and CA242 were 71.17%, 95.57% and 84.03%, respectively.
Conclusion:
1. Rabbit anti-human APE1 polyclonal antibody, with high titer and specificity, was successfully produced by a modified rapid immune procedure. This polyclonal antibody can be further applied in ELISA, Western blot and immunohistochemistry to elucidate the roles of APE1 protein played in many important cellular procedures.
2. Successfully established a sandwich ELISA assay for APE1 protein with good sensitivity and reproducibility for the first time.
3. Serum APE1 level in cancer patients was significantly increased compared to healthy. Serum APE1 combined with CEA, CA125 and CA242 can elevate the diagnostic sensitivity and correct rate, APE1 may become a new diagnostic biomarker for cancer.
4. Successfully established an indirect ELISA assay with good sensitivity and reproducibility for the first time. Serum APE1-Abs level of lung cancer patients was significantly higher than that of healthy. But variables such as sex, smoking status, TNM stages and histopathological types were not associated with APE1-Abs levels.
5. Serum APE1-Abs was also significantly correlated with response to therapy, and it may be a predictor of therapeutic efficacy.
6. APE1 antibody combined with CEA, CA125 and CA242 can elevate the diagnostic sensitivity and correct rate.
脱嘌呤/脱嘧啶核酸内切酶(APE1)是碱基切除修复(BER)途径中的一个限速酶,具有DNA损伤修复和氧化还原等多种功能,参与多种与细胞重要功能有关的反应,其功能异常可能导致肿瘤形成。研究表明,APE1基因表达具有器官和组织特异性,在肺癌组织中APE1表达水平较正常组织显著升高,且有明显的易位表达。APE1表达定位的改变及表达强度的变化可能是细胞表型异常的决定因素。由于肿瘤细胞增殖过快,不断有细胞发生凋亡和坏死,使细胞内大量蛋白质释放入血,血清中APE1蛋白含量可能升高。理论上,APE1表达水平和定位的改变会刺激机体产生抗APE1的自身抗体。目前认为血清抗体的产生是机体对肿瘤产生的一种特异性的免疫反应,间接反映了肿瘤的发展和演变。对APE1进行血清学检测可能有助于肿瘤的临床诊断,目前对APE1的检测大都为免疫组化或免疫印迹,其定位研究较为透彻,血清学检测在国内外尚未见报道。本研究首先制备兔抗人APE1多克隆抗体,然后建立改良的双抗体夹心ELISA和间接ELISA,检测血清APE1蛋白和抗体的表达,并探讨其与多肿瘤标志物联合检测在肺癌中的诊断作用及其临床应用价值。
研究目的
1.制备兔抗人APE1多克隆抗体,特异性亲和纯化并对其生物学特性进行鉴定;
2.建立双抗体夹心ELISA检测血清中APE1蛋白并结合蛋白芯片检测结果探讨其在肺癌诊断中的作用;
3.建立间接ELISA检测血清中APE1抗体并结合蛋白芯片检测结果探讨其临床应用价值。
研究内容和方法
1.兔抗人APE1多克隆抗血清的制备
用纯化的全长APE1蛋白免疫新西兰大白兔,采用一种快速免疫法制备APE1多克隆抗体,ELISA追踪检测抗体效价,Protein A亲和层析法纯化特异性IgG抗体,Western blot和免疫组化检测抗体的特性,为进一步建立双抗体夹心ELISA提供抗体。
2.改良的双抗体夹心ELISA检测血清APE1蛋白方法的建立
以鼠抗人APE1单克隆抗体包被高亲和力酶标板,APE1全长蛋白为标准品,兔抗人APE1多克隆抗体为检测抗体,HRP标记羊抗兔IgG为酶标抗体建立双抗体夹心ELISA,棋盘滴定法确定各抗体的最佳工作浓度并对ELISA方法的灵敏度、特异度和稳定性进行检测。采用所建立的ELISA方法检测肺癌患者和健康体检者血清并结合蛋白芯片检测结果进行统计学分析,探讨其临床意义。
3.间接ELISA检测血清APE1抗体方法的建立
以全长APE1蛋白包被高结合力酶标板,HRP标记羊抗人IgG为酶标抗体建立间接ELISA。采用所建立的ELISA方法检测肺癌患者和健康体检者血清并结合蛋白芯片检测结果进行统计学分析,从不同角度探讨其临床应用价值。
研究结果
1.成功制备了兔抗人APE1多克隆抗体。ELISA检测显示该抗体的效价达到1:128000,相对亲和力常数为8.96×10-6mol/L。Western blot和免疫组化显示该抗体能与APE1蛋白特异性结合。此外,该抗体还可用于小鼠和大鼠组织APE1蛋白的检测。
2.建立的夹心ELISA检测APE1蛋白的线性范围为8.0～200 ng/mL;批内变异系数为8.37%～8.97%,批间变异系数为10.47%～14.57%;平均回收率为90.83%。血清中APE1蛋白含量呈偏态分布,健康体检者和肺癌患者血清APE1含量95%可信区间分别为3.67ng/mL～16.58ng/mL和7.06ng/mL～24.90ng/mL,肺癌患者血清APE1蛋白水平明显高于健康体检者(P<0.05)。
3.回顾性分析发现,CEA、CA125和CA242三项标志物对肺癌诊断的灵敏度和准确率较高。APE1与这三项指标联合检测对肺癌诊断的灵敏度为68.57%,特异度为80.00%,准确率为74.15%。联合APE1检测较单一标志物检测的诊断灵敏度和准确率都有所提高,但是同时也降低了诊断的特异度。
4.建立的间接ELISA在血清稀释度为1:3200时仍能检测的到。批内变异系数平均为8.52%,批间变异系数平均为9.09%。肺癌患者血清APE1抗体水平明显高于健康体检者(P<0.05),APE1抗体阳性率分别为34.49%和3.43%,二者有显著性差异(P<0.05);但APE1抗体在不同性别,不同吸烟状态,不同TNM分期和不同组织病理学分型上并无显著性差异(P>0.05)。
5. APE1抗体还与治疗疗效紧密相关,化疗后APE1抗体水平显著高于治疗前;治疗有应答者(CR+PR)血清APE1抗体在化疗后比化疗前显著升高(P<0.05),而无应答者(SD+PD)血清APE1抗体水平在化疗前后没有显著性差别(P>0.05),提示血清APE1抗体可能对预测肺癌的化疗敏感性有重要参考价值。
6.血清APE1抗体与肺癌血清检测常用的肿瘤标志物CEA、CA125、和CA242联合检测的诊断灵敏度、特异度和准确率分别为71.17%、95.57%和84.03%。
结论
1.成功制备了兔抗人APE1多克隆抗体并进行了特异性亲和纯化,抗体效价和纯度均很高;Western blot和免疫组化显示此抗体特异性良好,可满足实验要求。
2.首次成功建立了改良的双抗体夹心ELISA检测APE1蛋白,所建立的ELISA方法的灵敏度、特异度、精确度、批内批间差异和稳定性均满足实验要求。
3.健康体检者和肺癌患者血清中均能检测到APE1蛋白,但表达量不同,肺癌患者血清APE1蛋白明显高于健康体检者;APE1与CEA、CA125、和CA242联合检测能提高诊断的准确率,提示APE1有望成为一种新的肿瘤标志物。
4.首次成功建立了间接ELISA检测血清APE1抗体,肺癌患者血清APE1抗体水平明显高于健康体检者,但APE1抗体在不同性别、吸烟状态、TNM分期和组织病理学分型上并无显著性差异。
5.血清APE1抗体还与治疗疗效紧密相关,血清APE1抗体可能对预测肺癌的化疗敏感性有重要参考价值。
6.血清APE1抗体与肺癌血清检测常用的肿瘤标志物CEA、CA125、和CA242联合检测可提高诊断的灵敏度和准确率。APE1抗体有可能成为肺癌辅助诊断的指标之一。
Lung cancer is the most common malignant tumor with high morbidity and mortality in China, and the morbidity is increasing year by year. The World Health Organization pointed out, 90~95 percent cancer is curable with early diagnosis and timely treatment. Early diagnosis of lung cancer is still the most effective way to improve the survival rate. Tumor markers have been used in the tumor screen, diagnosis, prognosis, evaluation of efficacy and the follow-up of high-risk groups. With recent advances in biotechnology such as proteomics, FISH, ELISA, RT-PCR, and SELDI-TOF, many promising biomarkers have been identified and are currently under investigation and validation. However, it is unlikely that any tumor marker will perfectly diagnose lung cancer. At present, the recombination of multiple predictive factors will be a realistic way to improve lung cancer in screening and staging. At present, searching for more specific and sensitive tumor markers is the most promising way to improve cancer diagnosis.
APE1 is a multifunctional protein that not only repairs the apurinic/apyrimidinic sites of DNA lesions, acting as a rate-limiting enzyme of BER pathway, but also functions as a reduction-oxidation factor protecting cells against the toxic effects. We believe the dysfunction of APE1 have the potential in increasing susceptibility of cancer. Most studies show that, lung cancer existed APE1 overexpression and altered localization, these alteration are likely to be the abnormal cell phenotype determinants. Since the tumor cell multiplication is excessively quickly, within unceasingly cell apoptosis and necrosis, a large number of cellular proteins released into the blood, so we proposed that APE1 protein in serum may be increased. APE1’s altered expression, primarily at the protein or subcellular localization level may stimulate organism to produce antibodies. As the antibody amount is more than the antigen and antibody exist in serum for a long time, so the detection of specific antibody may allow for identification of an underlying tumor. APE1 is expected to become a new biomarker for lung cancer.
To our knowledge, there is no report about APE1 proteins or antibodies level in serum. Studies on screening APE1 alterations were mostly by immunohistochemical staining or western blot. So far, this is the first examination of serum APE1 level in relation to cancer diagnosis. Our main objective of this prospective study is 1) to prepare rabbit anti-human APE1 polyclonal antibody 2) to evaluate clinical value of serum APE1 protein and antibody or in combination with CEA, CA125 and CA242 in distinguishing between lung cancer patients and healthy.
Objective:
1. To prepare rabbit anti-human APE1 polyclonal antibody and then characterize it.
2. To establish a sandwich ELISA for quantitative measurement of APE1 protein, measure serum APE1 level in healthy and lung cancer patients, and to study its clinical application in combination with CEA, CA125 and CA242.
3. To detect serum APE1 antibody in lung cancer patients and healthy by indirect ELISA, monitor patients before and after chemotherapy, combine with CEA, CA125 and CA242 to evaluate if serum APE1 antibody can be used as a new tumor marker and predictor of therapeutic efficacy.
Materials and Methods:
1. Preparation and characterization of rabbit anti-human APE1 polyclonal antibody Rabbit anti-human APE1 polyclonal antibody was prepared by a modified rapid immune procedure followed by purification of specific avidity column. The efficacy of this polyclonal antibody was certificated by ELISA, Western blot and immunohistochemistry.
2. Establishment and primary application of a sandwich ELISA for quantitative measurement of APE1
The assay was based on the sandwiching of the antigen between a monoclonal mouse anti-APE1 antibody, pre-coated on a 96 well polystyrene plate, and a polyclonal rabbit anti-APE1 antibody, which was then detected with a peroxidase-labeled goat anti-rabbit antibody. The optimal concentrations were defined by titration. Standard curve was performed using purified APE1 protein and was judged by sensitivity, reproducibility and recovery rate. The sandwich ELISA was tried to measure the APE1 levels in healthy and lung cancer patients, and then combined with CEA, CA125 and CA242 to study its clinical application.
3. Establishment and primary application of indirect ELISA in lung cancer and healthy Indirect ELISA was carried out to detect APE1-Abs in 345 lung cancer patients and 350 healthy. Ninety-one patients were monitored before and after chemotherapy. The data was analyzed by independent t-test, ROC curve and chi-square test. APE1 antibody was combined with CEA, CA125 and CA242 to study its clinical application.
Results:
1. The titer of the antiserum obtained in this experiment determined by ELISA was up to 1∶128 000.The kaff value of the antibody was 8.96×10-6mol/L. Western blot results showed the antibody has high specificity to APE1 protein. The final purified antibody was highly specific to native form of APE1 protein in human, mouse and rat.
2. In the range of 8.0 ng/mL to 200 ng/mL, the APE1 antigen showed a good linearity in the standard curve. The sensitivity of this assay was 2.0 ng/mL. The intra-assay precision and inter-assay precision were 8.37%～8.97% and 10.47%～14.57% respectively. The average recovery rate was 90.83 %. APE1 protein levels in serum were skewed distribution, The 95% CI of serum APE1 concentration in healthy and lung cancer patients were 3.67ng/mL～16.58ng/mL and 7.06ng/mL～24.90ng/mL respectively. The results demonstrated a significant increase of serum APE1 level in cancer patients compared to healthy (P<0.05).
3. Retrospective analysis found that, CEA, CA125 and CA242 were more specific for diagnosis of lung cancer and had higher positive rate. APE1 combined with these three makers can elevate the diagnostic sensitivity and correct rate. In this study, the diagnostic sensitivity, specificity and correct rate were 68.57%, 80.00% and 74.15%, respectively. The diagnostic sensitivity and accuracy were improved than a single indicator, but at the same time the specificity of diagnosis were reduced.
4. Serum APE1-Abs level of lung cancer patients was significantly higher than that of healthy volunteers (P=0.000). For each possible cut-point, the resulting sensitivity and specificity were indicated as a point on the ROC graph. The area under ROC was 0.735. One hundred and nineteen (34.49%) lung cancer patients and 12 (3.43%) healthy were positive with APE1-Abs. But variables such as sex, smoking status, TNM stages and histopathological types were not associated with APE1-Abs (P>0.05, respectively).
5. We also found serum APE1-Abs were significantly associated with response to therapy. Serum APE1-Abs of responders were significantly higher after chemotherapy (P=0.000), while APE1-Abs level in non-responders, the difference was not significant (P=0.393).
6. Diagnostic sensitivity, specificity and correct rate of APE1 antibody with combination of CEA, CA125 and CA242 were 71.17%, 95.57% and 84.03%, respectively.
Conclusion:
1. Rabbit anti-human APE1 polyclonal antibody, with high titer and specificity, was successfully produced by a modified rapid immune procedure. This polyclonal antibody can be further applied in ELISA, Western blot and immunohistochemistry to elucidate the roles of APE1 protein played in many important cellular procedures.
2. Successfully established a sandwich ELISA assay for APE1 protein with good sensitivity and reproducibility for the first time.
3. Serum APE1 level in cancer patients was significantly increased compared to healthy. Serum APE1 combined with CEA, CA125 and CA242 can elevate the diagnostic sensitivity and correct rate, APE1 may become a new diagnostic biomarker for cancer.
4. Successfully established an indirect ELISA assay with good sensitivity and reproducibility for the first time. Serum APE1-Abs level of lung cancer patients was significantly higher than that of healthy. But variables such as sex, smoking status, TNM stages and histopathological types were not associated with APE1-Abs levels.
5. Serum APE1-Abs was also significantly correlated with response to therapy, and it may be a predictor of therapeutic efficacy.
6. APE1 antibody combined with CEA, CA125 and CA242 can elevate the diagnostic sensitivity and correct rate.
引文
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[2] Fritz G, Gr?sch S, Tomicic M, Kaina B. APE /Ref-1 and the mammalian response to genotoxic stress [J]. Toxicology. 2003; 93 (1-2): 67–78.
[3] Evans AR, Limp-Foster M, Kelley MR. Going APE over ref-1 [J]. Mutat Res. 2000; 461 (2): 83-108.
[4] Barzilay G, Hickson ID. Structure and function of apurinic/apyrimidinic endonucleases [J]. Bioessays. 1995; 17 (8): 713-9.
[5] Sander M, Lowenhaupt K, Rich A. Drosphila Rrp1 protein: an apurinic endonuclease with homologous recombination activities [J]. Proc Natl Acad Sci USA. 1991; 88 (15): 6780-4.
[6] Yoon JH, Singh P, Lee DH, et al. Characterization of the 3' --> 5' exonuclease activity found in human nucleoside diphosphate kinase 1 (NDK1) and several of its homologues [J]. Biochemistry. 2005; 44 (48): 15774-86.
[7] Purohit S, Arenaz P. Molecular cloning, sequence and structure analysis of hamster apurinic/ apyrimidinic endonuclease (chAPE1) gene [J]. Mutat Res. 1999; 435 (3): 215-24.
[8] Hu YX, Guo JY, Shen L, et al. Get effective polyclonal antiserum in one month [J].Cell Res, 2002, 12(2): 157-160.
[9]哈洛E,莱恩D.抗体技术实验指南[M].北京科学出版社;2002: 21-23.
[10] Beatty JD, Barbara BG, William GV. Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay [J]. J Immunol Methods. 1987; 100 (1-2): 173-9.
[11] Parsons JL, Dianova II, Dianov GL. APE1 is the major 3’phosphoglycolate activity in human cell extracts [J]. Nucleic Acids Res. 2004; 32 (12): 3531 - 6.
[12] Demple B, Sung JS. Molecular and biological roles of Ape1 protein in mammalian base excision repair [J]. DNA Repair (Amst). 2005; 4 (12): 1442-9.
[13] Wang D, Demp le B. Modulation of the 5′deoxyribose-5-phosphatelyase and DNA synthesis activities of mammalian DNA polymerase beta by apurinic/apyrimidinicendonuclease 1 [J]. J Biol Chem. 2004; 279 (24): 25268-75.
[14]张云嵩,范士志,王东等. APE1在非小细胞肺癌中的表达特点及其与预后的关系[J].第三军医大学学报. 2007; 29 (9): 776-8.
[15] Duguid JR, Eble JN, Wilson TM, Kelley MR. Differential cellular and subcellular expression of the human multifunctional apurinic/apyrimidinic endonuclease (APE/Ref-1) DNA repair enzyme [J]. Cancer Res. 1995; 55 (24): 6097–102.
[16] Tell G, Damante G, Caldwell D, Kelley MR. The intracellular localization of APE1/Ref-1: More than a passive phenomenon? [J]. Antioxid Redox Signal. 2005; 7 (3-4): 367–84.
[17] Puglisi F, Barbone F, Tell G, et a1.Prognostic role of APEl subcellar expression in stage I-Ⅲbreast carcinomas [J]. Oncol Rep. 2002; 9 (1): 11-14.
[18] Futagami S, Kawagoe T, Horie A, Shindo T. Celecoxib inhibits apurinic/apyrimidinic endonuclease-1 expression and prevents gastric cancer in Helicobacter pylori-infected mongolian gerbils [J]. Digestion. 2008; 78 (2-3): 93-102.
[19] Moore DH, Michael H, Tritt R, et al. Alterations in the expression of the DNA repair/redox enzyme APE/ref-1 in epithelial ovarian cancers [J]. Clin Cancer Res. 2000; 6 (2): 602-9.
[20] Nielsen UB, Geierstanger BH. Multiplexed sandwich assays in microarray format [J]. J Immunol Methods. 2000; 290 (1-2): 107-20.
[21] Gonzalez RM, Seurynck-Servoss SL, Crowley SA, et al. Development and validation of sandwich ELISA microarrays with minimal assay interference [J]. J Proteome Res. 2008; 7 (6): 2406-14.
[22] Shu-Ling L, Daniel W. Enzymes and related proteins as cancer biomarkers: A proteomic approach [J]. Clinica Chimica Acta. 2007; 381 (2): 93-7.
[23] Wang D, Xiang DB, Yang XQ, et al. APE1 overexpression is associated with cisplatin resistance in non-small cell lung cancer and targeted inhibition of APE1 enhances the activity of cisplatin in A549 cells [J]. Lung Cancer. 2009; Mar 24. [Epub ahead of print]
[24] Xiang DB, Chen ZT, Wang D, et al. Chimeric adenoviral vector Ad5/F35-mediated APE1 siRNA enhances sensitivity of human colorectal cancer cells to radiotherapy in vitro and in vivo [J]. Cancer Gene Ther. 2008; 15 (10): 625-35.
[25] Tanner B, Grimme S, Schiffer I, et al. Nuclear expression of apurinic/ apyrimidinic endonuclease increases with progression of ovarian carcinomas [J]. Gynecologic Oncology. 2004; 92 (2): 568-77.
[26] Binder SR. Autoantibody detection using multiplex technologies [J]. Lupus. 2006; 15 (7): 412-21.
[27] S?reide K. Receiver-operating characteristic curve analysis in diagnostic, prognostic and predictive biomarker research [J]. J Clin Pathol. 2009; 62 (1): 1-5.
[28] Roman S, Mehta P, Sosa JA. Medullary thyroid cancer: early detection and novel treatments. [J]. Curr Opin Oncol. 2009; 21 (1): 5-10.
[29] Hoebena A, Landuyt B, Botrus G, et al. Proteomics in cancer research: Methods and application of array-based protein profiling technologies [J]. Analytica Chimica Acta. 2006; 564 (1): 19-33.
[30] Chen F, Li WM, Wang DM, et al. Clinical value of combined detection of serum tumor markers in lung cancer diagnosis [J]. Sichuan Da Xue Xue Bao Yi Xue Ban. 2008; 39 (5): 832-5.
[31] Novellino L, Castelli C, Parmiani G. A listing of human tumor antigens recognized by T cells: March 2004 update [J]. Cancer Immunol Immunother. 2005; 54 (3): 187-207.
[32] Biagini RE, Sammons DL, Smith JP, et al. Rapid, sensitive, and specific lateral-flow immunochromatographic device to measure anti-anthrax protective antigen immunoglobulin g in serum and whole blood [J]. Clin Vaccine Immunol. 2006; 13 (5): 541-6.
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