血紅蛋白HbA2與HbF免疫學(xué)檢測體系的建立與初步應(yīng)用
發(fā)布時間：2018-07-21 21:10
【摘要】：人類血紅蛋白鏈可分為α鏈(α,ζ)與非α鏈(β,γ,,δ,ε)。正常成人血紅蛋白的主要成份是血紅蛋白A(HbA),占全部血紅蛋白的96.5-97.5%,由一對α鏈及一對β鏈(α2β2)構(gòu)成；其余成分為血紅蛋白A2(HbA2),由α2δ2構(gòu)成,占2.5-3.5%,而胎兒血紅蛋白即HbF(α-2γ2),占1%以下。在一些血液系統(tǒng)疾病中,包括α與β地中海貧血及其基因攜帶者的血紅蛋白比例有所改變。 β地中海貧血(以下簡稱β地貧)是由于β珠蛋白基因缺陷導(dǎo)致β珠蛋白鏈合成減少或缺如所引起的遺傳性血液病,其典型的臨床表現(xiàn)是小細(xì)胞低色素性貧血[1-3]。該病廣泛分布于世界許多地區(qū),東南亞是高發(fā)區(qū)之一,在我國南方地區(qū)如廣東、廣西、海南的發(fā)病率和攜帶率都很高。重癥地貧發(fā)病率在廣東省的出生缺陷中排位第二,以α和β地貧基因攜帶率分別為8.53%和2.54%(合計達(dá)11.07%)估算,全省每年生育重癥α地貧(Bart's水腫胎和Hb H病)和重癥β地貧患兒的高風(fēng)險育齡夫婦可達(dá)4,000對以上[1-5]。重癥β地貧患兒主要依靠頻繁的輸血維持生命,且多在未成年前天折,給社會和家庭在經(jīng)濟(jì)上和精神上帶來沉重的負(fù)擔(dān)。因此,通過檢測β地貧從而進(jìn)行產(chǎn)前診斷是阻止該病重癥患兒出生最為有效的措施。 產(chǎn)前診斷是目前國際上公認(rèn)的預(yù)防地貧的首選措施。通過產(chǎn)前診斷可有效預(yù)防患兒的出生,從而達(dá)到預(yù)防重癥地貧患兒出生的目的。在人群中大規(guī)模篩查p地貧基因攜帶者是實現(xiàn)地貧預(yù)防的必要步驟。基于中國南方p地貧發(fā)生的人群高頻率,開展大規(guī)模遺傳病群體預(yù)防具有重要意義。廣大的育齡期青年和孕期夫婦可作為人群篩查的主要對象。 傳統(tǒng)用于檢測β地貧攜帶者的方法有表型篩查法和基因檢測兩大類。表型篩查法主要是血液學(xué)檢查和血紅蛋白分析,如紅細(xì)胞形態(tài)、紅細(xì)胞指數(shù)、紅細(xì)胞滲透脆性、血紅蛋白理化性質(zhì)測定、血紅蛋白電泳等幾個方面,但這些方法變異范圍大、準(zhǔn)確性低。近年采用的高效液相色譜法,由于儀器設(shè)備昂貴,不適用于廣大地貧高發(fā)區(qū)的高通量篩查。基因檢測法,是從樣品的制備、目的基因的PCR擴(kuò)增、PCR產(chǎn)物的鑒定和分析到反向點雜交實驗(RDB)對地貧的基因分型。用上述篩查后進(jìn)行基因診斷確診的分析策略是地貧診斷的傳統(tǒng)技術(shù)路線[6-9]。該方法準(zhǔn)確、可靠,但操作過程較為繁雜,難以實現(xiàn)快速和高通量,且檢測費用較高,不易在基層醫(yī)院普及,從而限制了地貧基因攜帶者的大規(guī)模篩查。因此研究和開發(fā)具有高通量、廉價、易于在基層使用的新技術(shù)勢在必行。 p地貧患者及基因攜帶者的p珠蛋白鏈合成障礙導(dǎo)致HbA2水平發(fā)生改變,其外周血紅細(xì)胞HbA2含量可高出正常人一倍。因此,HbA2的免疫學(xué)檢測可作為β地貧的篩查指標(biāo)；同時,β地貧患者及其基因攜帶者外周血中HbF亦普遍升高,因此HbF定量可作為β地貧篩查的輔助指標(biāo),二者聯(lián)合應(yīng)用,可以完善對地貧的免疫學(xué)篩查、診斷及預(yù)后判斷。ELISA法具有特異性好、敏感度高、操作簡便等優(yōu)點,可進(jìn)行高通量檢測,適用于大規(guī)模篩查。本科室在成功研制了檢測α地貧免疫學(xué)試劑盒的基礎(chǔ)上,又成功制備了可分別特異性識別HbA2、HbF的單克隆抗體,可用于p地貧的大規(guī)模篩查、輔助診斷以及p地貧患者的病情監(jiān)測與預(yù)后判斷。 據(jù)文獻(xiàn)報道,胎兒血紅蛋白作為一種胚胎期蛋白與惡性腫瘤有一定關(guān)聯(lián),在一些惡性腫瘤如兒童急性白血病、結(jié)腸癌、生殖細(xì)胞腫瘤中HbF含量出現(xiàn)再次增高[10-14],本文通過流式細(xì)胞術(shù)、PCR技術(shù)、免疫組化的方法,檢測腫瘤細(xì)胞中HbF的表達(dá)情況。 在前期單抗的制備工作中,我們成功研制了4株血紅蛋白單抗,其中2H4、1H11特異性針對HbA2,即Hbδ鏈特異性；2C9、1E1O則具有HbA(Hbβ)HbA2雙特異性。通過交叉配對選擇合適的抗體對,確定了以單抗2H4為捕獲抗體,2C9為檢測抗體并直接標(biāo)記辣根過氧化物酶,進(jìn)行了最佳工作濃度、反應(yīng)時間及溫度的選擇與確定。確定單抗2H4最佳包被濃度為2ug/ml,HRP-2C9最佳檢測濃度為1μg/ml。通過比較不同的血液標(biāo)本保存條件、不同的封閉條件以及樣品稀釋液對體系的作用,同時對樣品裂解緩沖液與裂解條件進(jìn)行了摸索,對體系進(jìn)一步優(yōu)化。經(jīng)鑒定該體系可特異性結(jié)合HbA2,而與HbA、HbF及Hbzeta鏈無交叉反應(yīng)；其敏感度可達(dá)ng/ml水平,考慮臨床應(yīng)用的方便,我們降低了檢出敏感性以減少稀釋倍數(shù)高可能形成的誤差。所獲HbA2標(biāo)準(zhǔn)曲線擬合關(guān)系較好,能夠特異、敏感地檢測p地貧基因攜帶者及正常人血中的HbA2。此外,我們還正在嘗試用同樣的雙單抗建立時間分辨免疫熒光檢測體系,以提高在一定濃度范圍內(nèi)的分辨率。 基于前期實驗工作所獲得的三株抗HbF單抗與親和純化兔抗HbF多克隆抗體進(jìn)行組合,證明以單抗2C8作為捕獲抗體相對其他單抗可獲得更好的靈敏度,且本底也比較低。同時,我們對單多抗雙夾心ELISA的包被抗體濃度、檢測抗體濃度、包被緩沖液、封閉液及樣品稀釋液等條件予以優(yōu)化。尤其是采用了不同于一般雙夾心ELISA的Tris-HCI(pH8.0)緩沖體系,而多數(shù)ELISA均采用PBS體系。同時對體系的特異性、靈敏度、檢測范圍及批間批內(nèi)穩(wěn)定性進(jìn)行鑒定。并用所確立條件初步測定了經(jīng)高效液相色譜法檢測的100份外周血樣品。結(jié)果表明：本檢測體系特異性好,與HbA2和HbA無交叉反應(yīng)。其檢測靈敏度約為0.039ug/ml,檢測范圍0.039-24.66ug/ml,重復(fù)性好；100份血標(biāo)本的檢測結(jié)果與HPLC的檢測結(jié)果有良好的相關(guān)性。 鑒于一些胚胎抗原可在若干腫瘤細(xì)胞及血清中表達(dá),并由此被作為腫瘤相關(guān)抗原的標(biāo)志物,我們以FITC標(biāo)記前期制備的HbF單抗,用流式細(xì)胞術(shù)檢測了13種腫瘤細(xì)胞系(K562、HL60等)中胎兒血紅蛋白的表達(dá)情況。同時提取腫瘤細(xì)胞系的RNA,檢測胎兒血紅蛋白在基因水平的表達(dá)情況。結(jié)果表明,除了K562細(xì)胞外,其余腫瘤細(xì)胞系均不表達(dá)胎兒血紅蛋白。K562是紅-白血病細(xì)胞系,具有向紅系或白系分化的趨勢,因此,K562可表達(dá)胎兒血紅蛋白；其他腫瘤細(xì)胞系,如血液系統(tǒng)的腫瘤細(xì)胞系HL-60,及文獻(xiàn)報道出現(xiàn)原代腫瘤細(xì)胞表達(dá)HbF的生殖細(xì)胞腫瘤、腸道的惡性腫瘤,均未檢出胎兒血紅蛋白的表達(dá)。原代腫瘤組織切片的免疫組化結(jié)果中,可在肝細(xì)胞肝癌、宮頸癌、腹膜B細(xì)胞淋巴瘤的腫瘤組織微血管內(nèi)發(fā)現(xiàn)有HbF表達(dá)陽性的細(xì)胞,但在腫瘤細(xì)胞和組織未能發(fā)現(xiàn)有HbF表達(dá)。
[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 偽 chains and a pair of beta chains ( 偽2尾2 ) .
The other components are hemoglobin A2 ( HbA2 ) , which is composed of 偽2未2 , which accounts for 2.5 - 3.5 % , and fetal hemoglobin , HbF ( 偽 - 2緯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 偽 - 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 .
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392
本文編號：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 偽 chains and a pair of beta chains ( 偽2尾2 ) .
The other components are hemoglobin A2 ( HbA2 ) , which is composed of 偽2未2 , which accounts for 2.5 - 3.5 % , and fetal hemoglobin , HbF ( 偽 - 2緯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 偽 - 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 .
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392
【參考文獻(xiàn)】
相關(guān)期刊論文 前5條
1 吳潔瑩,廖燦,李堅,黃以寧;多重PCR用于缺失型α-地中海貧血的基因檢測[J];中國實驗血液學(xué)雜志;2004年04期
2 周玉球,張永良,李莉艷,李文典,莫秋華,鄭勤,徐湘民;單管多重PCR快速檢測中國人三種常見缺失型α-地中海貧血基因[J];中華醫(yī)學(xué)遺傳學(xué)雜志;2005年02期
3 江海燕,劉艷君,朱平,韓強(qiáng)濤,富寧;人可溶性CD14檢測體系的建立[J];中國實驗診斷學(xué);2004年01期
4 程偉;劉艷君;陳遠(yuǎn)東;鐘梅;富寧;;抗胎兒血紅蛋白單克隆抗體的制備及特性鑒定[J];中國實驗診斷學(xué);2009年02期
5 程偉;富寧;;人胎兒血紅蛋白的檢測及其臨床意義[J];中國實驗診斷學(xué);2009年10期
相關(guān)碩士學(xué)位論文 前1條
1 孫曼娜;中國南方廣西壯族自治區(qū)血紅蛋白病的分子流行病學(xué)調(diào)查[D];南方醫(yī)科大學(xué);2010年
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