血红蛋白HbA2与HbF免疫学检测体系的建立与初步应用
发布时间：2018-07-21 21:10
【摘要】：人类血红蛋白链可分为α链(α,ζ)与非α链(β,γ,,δ,ε)。正常成人血红蛋白的主要成份是血红蛋白A(HbA),占全部血红蛋白的96.5-97.5%,由一对α链及一对β链(α2β2)构成；其余成分为血红蛋白A2(HbA2),由α2δ2构成,占2.5-3.5%,而胎儿血红蛋白即HbF(α-2γ2),占1%以下。在一些血液系统疾病中,包括α与β地中海贫血及其基因携带者的血红蛋白比例有所改变。 β地中海贫血(以下简称β地贫)是由于β珠蛋白基因缺陷导致β珠蛋白链合成减少或缺如所引起的遗传性血液病,其典型的临床表现是小细胞低色素性贫血[1-3]。该病广泛分布于世界许多地区,东南亚是高发区之一,在我国南方地区如广东、广西、海南的发病率和携带率都很高。重症地贫发病率在广东省的出生缺陷中排位第二,以α和β地贫基因携带率分别为8.53%和2.54%(合计达11.07%)估算,全省每年生育重症α地贫(Bart's水肿胎和Hb H病)和重症β地贫患儿的高风险育龄夫妇可达4,000对以上[1-5]。重症β地贫患儿主要依靠频繁的输血维持生命,且多在未成年前天折,给社会和家庭在经济上和精神上带来沉重的负担。因此,通过检测β地贫从而进行产前诊断是阻止该病重症患儿出生最为有效的措施。 产前诊断是目前国际上公认的预防地贫的首选措施。通过产前诊断可有效预防患儿的出生,从而达到预防重症地贫患儿出生的目的。在人群中大规模筛查p地贫基因携带者是实现地贫预防的必要步骤。基于中国南方p地贫发生的人群高频率,开展大规模遗传病群体预防具有重要意义。广大的育龄期青年和孕期夫妇可作为人群筛查的主要对象。 传统用于检测β地贫携带者的方法有表型筛查法和基因检测两大类。表型筛查法主要是血液学检查和血红蛋白分析,如红细胞形态、红细胞指数、红细胞渗透脆性、血红蛋白理化性质测定、血红蛋白电泳等几个方面,但这些方法变异范围大、准确性低。近年采用的高效液相色谱法,由于仪器设备昂贵,不适用于广大地贫高发区的高通量筛查。基因检测法,是从样品的制备、目的基因的PCR扩增、PCR产物的鉴定和分析到反向点杂交实验(RDB)对地贫的基因分型。用上述筛查后进行基因诊断确诊的分析策略是地贫诊断的传统技术路线[6-9]。该方法准确、可靠,但操作过程较为繁杂,难以实现快速和高通量,且检测费用较高,不易在基层医院普及,从而限制了地贫基因携带者的大规模筛查。因此研究和开发具有高通量、廉价、易于在基层使用的新技术势在必行。 p地贫患者及基因携带者的p珠蛋白链合成障碍导致HbA2水平发生改变,其外周血红细胞HbA2含量可高出正常人一倍。因此,HbA2的免疫学检测可作为β地贫的筛查指标；同时,β地贫患者及其基因携带者外周血中HbF亦普遍升高,因此HbF定量可作为β地贫筛查的辅助指标,二者联合应用,可以完善对地贫的免疫学筛查、诊断及预后判断。ELISA法具有特异性好、敏感度高、操作简便等优点,可进行高通量检测,适用于大规模筛查。本科室在成功研制了检测α地贫免疫学试剂盒的基础上,又成功制备了可分别特异性识别HbA2、HbF的单克隆抗体,可用于p地贫的大规模筛查、辅助诊断以及p地贫患者的病情监测与预后判断。 据文献报道,胎儿血红蛋白作为一种胚胎期蛋白与恶性肿瘤有一定关联,在一些恶性肿瘤如儿童急性白血病、结肠癌、生殖细胞肿瘤中HbF含量出现再次增高[10-14],本文通过流式细胞术、PCR技术、免疫组化的方法,检测肿瘤细胞中HbF的表达情况。 在前期单抗的制备工作中,我们成功研制了4株血红蛋白单抗,其中2H4、1H11特异性针对HbA2,即Hbδ链特异性；2C9、1E1O则具有HbA(Hbβ)HbA2双特异性。通过交叉配对选择合适的抗体对,确定了以单抗2H4为捕获抗体,2C9为检测抗体并直接标记辣根过氧化物酶,进行了最佳工作浓度、反应时间及温度的选择与确定。确定单抗2H4最佳包被浓度为2ug/ml,HRP-2C9最佳检测浓度为1μg/ml。通过比较不同的血液标本保存条件、不同的封闭条件以及样品稀释液对体系的作用,同时对样品裂解缓冲液与裂解条件进行了摸索,对体系进一步优化。经鉴定该体系可特异性结合HbA2,而与HbA、HbF及Hbzeta链无交叉反应；其敏感度可达ng/ml水平,考虑临床应用的方便,我们降低了检出敏感性以减少稀释倍数高可能形成的误差。所获HbA2标准曲线拟合关系较好,能够特异、敏感地检测p地贫基因携带者及正常人血中的HbA2。此外,我们还正在尝试用同样的双单抗建立时间分辨免疫荧光检测体系,以提高在一定浓度范围内的分辨率。 基于前期实验工作所获得的三株抗HbF单抗与亲和纯化兔抗HbF多克隆抗体进行组合,证明以单抗2C8作为捕获抗体相对其他单抗可获得更好的灵敏度,且本底也比较低。同时,我们对单多抗双夹心ELISA的包被抗体浓度、检测抗体浓度、包被缓冲液、封闭液及样品稀释液等条件予以优化。尤其是采用了不同于一般双夹心ELISA的Tris-HCI(pH8.0)缓冲体系,而多数ELISA均采用PBS体系。同时对体系的特异性、灵敏度、检测范围及批间批内稳定性进行鉴定。并用所确立条件初步测定了经高效液相色谱法检测的100份外周血样品。结果表明：本检测体系特异性好,与HbA2和HbA无交叉反应。其检测灵敏度约为0.039ug/ml,检测范围0.039-24.66ug/ml,重复性好；100份血标本的检测结果与HPLC的检测结果有良好的相关性。 鉴于一些胚胎抗原可在若干肿瘤细胞及血清中表达,并由此被作为肿瘤相关抗原的标志物,我们以FITC标记前期制备的HbF单抗,用流式细胞术检测了13种肿瘤细胞系(K562、HL60等)中胎儿血红蛋白的表达情况。同时提取肿瘤细胞系的RNA,检测胎儿血红蛋白在基因水平的表达情况。结果表明,除了K562细胞外,其余肿瘤细胞系均不表达胎儿血红蛋白。K562是红-白血病细胞系,具有向红系或白系分化的趋势,因此,K562可表达胎儿血红蛋白；其他肿瘤细胞系,如血液系统的肿瘤细胞系HL-60,及文献报道出现原代肿瘤细胞表达HbF的生殖细胞肿瘤、肠道的恶性肿瘤,均未检出胎儿血红蛋白的表达。原代肿瘤组织切片的免疫组化结果中,可在肝细胞肝癌、宫颈癌、腹膜B细胞淋巴瘤的肿瘤组织微血管内发现有HbF表达阳性的细胞,但在肿瘤细胞和组织未能发现有HbF表达。
[Abstract]:The human hemoglobin chain can be divided into alpha chain ( . alpha . , zeta ) and non - alpha chain ( . beta . , gamma , ,未 , 蔚 ) . Hemoglobin A ( HbA ) is the main component of normal adult hemoglobin , which accounts for 96.5 - 97.5 % of all the hemoglobin , and consists of a pair of 伪 chains and a pair of beta chains ( 伪2尾2 ) .
The other components are hemoglobin A2 ( HbA2 ) , which is composed of 伪2未2 , which accounts for 2.5 - 3.5 % , and fetal hemoglobin , HbF ( 伪 - 2纬2 ) , accounts for less than 1 % . In some blood system diseases , the ratio of hemoglobin to 尾 - thalassaemia and its gene carrier is changed .
尾 - globin gene deficiency results in the decrease of 尾 - globin chain and the loss of 尾 - globin chain . Its typical clinical manifestations are low - cell low - color anemia , which is widely distributed in many parts of the world .
The prenatal diagnosis is the first choice for prevention of extreme poverty in the world . It is necessary to prevent the birth of children with severe poverty by prenatal diagnosis . The large - scale screening of p - poor gene carriers in the population is necessary to realize the prevention of the disease .
This method is accurate and reliable , but it is not easy to be popularized in basic hospitals because of its high flux , low cost and easy to use in grass - roots hospitals .
P - globin chain synthesis in patients with p - poor patients and carriers resulted in a change in HbA2 level , and the HbA2 content in peripheral blood could be twice as high as that of normal persons . Therefore , the immunological detection of HbA2 could be used as a screening index for 尾 - site poor .
At the same time , the HbF in peripheral blood of patients with 尾 - poor and its gene carriers is also generally increased , so HbF can be used as an auxiliary index for screening of 尾 - site - poor . It can be used for high - throughput screening . It is suitable for large - scale screening . It has been successfully developed to test 伪 - lean immunoassay kit . It has been successfully developed to identify the monoclonal antibodies of HbA2 and HbF . It can be used for the large - scale screening , auxiliary diagnosis and prognosis of p - poor patients .
The expression of HbF in tumor cells was detected by flow cytometry , PCR and immunohistochemistry .
In the preparation of monoclonal antibody , four strains of hemoglobin were successfully developed . Among them , 2H4 and H11were specific for HbA2 , i.e . Hb 未 chain specificity .
The optimal concentration of monoclonal antibody 2H4 was 2ug / ml , the optimal concentration of HRP - 2C9 was 1 渭g / ml . The optimal concentration of McAb 2H4 was 2ug / ml , the optimal concentration of HRP - 2C9 was 1 渭g / ml .
The sensitivity can be up to ng / ml . Considering the convenience of clinical application , we have reduced the detection sensitivity to reduce the error of high dilution multiple . The HbA2 standard curve fitting relationship is good . It can detect the HbA2 in the blood of the carrier and normal blood of the p - poor gene . In addition , we are trying to establish the time - resolved immunofluorescence detection system with the same double monoclonal antibody to improve the resolution within a certain concentration range .
Three strains of anti - HbF monoclonal antibody against HbF monoclonal antibody and affinity purified rabbit anti - HbF polyclonal antibody were combined to prove that the monoclonal antibody 2C8 was used as the capture antibody to obtain better sensitivity than other monoclonal antibodies , and the specificity , sensitivity , detection range and the stability of the batch were optimized . The results showed that the specificity of the system was good , and the detection range was 0.039 - 24.66ug / ml , and the repeatability was good .
There was a good correlation between the results of detection of 100 blood samples and the results of HPLC .
In view of the expression of fetal hemoglobin in several tumor cells and serum , the expression of fetal hemoglobin in 13 tumor cell lines ( K562 , HL 60 , etc . ) was detected by flow cytometry . The results showed that fetal hemoglobin was not expressed in the other tumor cell lines except for K562 cells .
Other tumor cell lines , such as tumor cell lines HL - 60 of the blood system , and literature reports that the expression of HbF expression in primary tumor cells and intestinal malignant tumors were not detected . In the immunohistochemistry of primary tumor tissue sections , it was found that HbF expression was positive in hepatocellular carcinoma , cervical cancer , and peritoneal B - cell lymphoma , but HbF expression was not found in tumor cells and tissues .
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
本文编号：2136865
[Abstract]:The human hemoglobin chain can be divided into alpha chain ( . alpha . , zeta ) and non - alpha chain ( . beta . , gamma , ,未 , 蔚 ) . Hemoglobin A ( HbA ) is the main component of normal adult hemoglobin , which accounts for 96.5 - 97.5 % of all the hemoglobin , and consists of a pair of 伪 chains and a pair of beta chains ( 伪2尾2 ) .
The other components are hemoglobin A2 ( HbA2 ) , which is composed of 伪2未2 , which accounts for 2.5 - 3.5 % , and fetal hemoglobin , HbF ( 伪 - 2纬2 ) , accounts for less than 1 % . In some blood system diseases , the ratio of hemoglobin to 尾 - thalassaemia and its gene carrier is changed .
尾 - globin gene deficiency results in the decrease of 尾 - globin chain and the loss of 尾 - globin chain . Its typical clinical manifestations are low - cell low - color anemia , which is widely distributed in many parts of the world .
The prenatal diagnosis is the first choice for prevention of extreme poverty in the world . It is necessary to prevent the birth of children with severe poverty by prenatal diagnosis . The large - scale screening of p - poor gene carriers in the population is necessary to realize the prevention of the disease .
This method is accurate and reliable , but it is not easy to be popularized in basic hospitals because of its high flux , low cost and easy to use in grass - roots hospitals .
P - globin chain synthesis in patients with p - poor patients and carriers resulted in a change in HbA2 level , and the HbA2 content in peripheral blood could be twice as high as that of normal persons . Therefore , the immunological detection of HbA2 could be used as a screening index for 尾 - site poor .
At the same time , the HbF in peripheral blood of patients with 尾 - poor and its gene carriers is also generally increased , so HbF can be used as an auxiliary index for screening of 尾 - site - poor . It can be used for high - throughput screening . It is suitable for large - scale screening . It has been successfully developed to test 伪 - lean immunoassay kit . It has been successfully developed to identify the monoclonal antibodies of HbA2 and HbF . It can be used for the large - scale screening , auxiliary diagnosis and prognosis of p - poor patients .
The expression of HbF in tumor cells was detected by flow cytometry , PCR and immunohistochemistry .
In the preparation of monoclonal antibody , four strains of hemoglobin were successfully developed . Among them , 2H4 and H11were specific for HbA2 , i.e . Hb 未 chain specificity .
The optimal concentration of monoclonal antibody 2H4 was 2ug / ml , the optimal concentration of HRP - 2C9 was 1 渭g / ml . The optimal concentration of McAb 2H4 was 2ug / ml , the optimal concentration of HRP - 2C9 was 1 渭g / ml .
The sensitivity can be up to ng / ml . Considering the convenience of clinical application , we have reduced the detection sensitivity to reduce the error of high dilution multiple . The HbA2 standard curve fitting relationship is good . It can detect the HbA2 in the blood of the carrier and normal blood of the p - poor gene . In addition , we are trying to establish the time - resolved immunofluorescence detection system with the same double monoclonal antibody to improve the resolution within a certain concentration range .
Three strains of anti - HbF monoclonal antibody against HbF monoclonal antibody and affinity purified rabbit anti - HbF polyclonal antibody were combined to prove that the monoclonal antibody 2C8 was used as the capture antibody to obtain better sensitivity than other monoclonal antibodies , and the specificity , sensitivity , detection range and the stability of the batch were optimized . The results showed that the specificity of the system was good , and the detection range was 0.039 - 24.66ug / ml , and the repeatability was good .
There was a good correlation between the results of detection of 100 blood samples and the results of HPLC .
In view of the expression of fetal hemoglobin in several tumor cells and serum , the expression of fetal hemoglobin in 13 tumor cell lines ( K562 , HL 60 , etc . ) was detected by flow cytometry . The results showed that fetal hemoglobin was not expressed in the other tumor cell lines except for K562 cells .
Other tumor cell lines , such as tumor cell lines HL - 60 of the blood system , and literature reports that the expression of HbF expression in primary tumor cells and intestinal malignant tumors were not detected . In the immunohistochemistry of primary tumor tissue sections , it was found that HbF expression was positive in hepatocellular carcinoma , cervical cancer , and peritoneal B - cell lymphoma , but HbF expression was not found in tumor cells and tissues .
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
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