目的 利用多肽合成技术制备抗胎儿血红蛋白(fetal hem-oglobin,HBF)γ链的抗体,探讨其用于检测孕妇外周血中胎儿有核红细胞(nucleated red blood cell,NRBC)进行无创性产前基因诊断的可行性.方法 针对胎儿血红蛋白的特异性抗原表位,选定第69～78位HbF-γ特异的11个氨基酸残基的肽段为免疫原,将人工合成的胎儿血红蛋白γ链的多肽与载体蛋白(KLH)偶联,佐剂乳化后免疫羊,制备羊抗人胎儿血红蛋白的抗血清,经蛋白G纯化,HbF特异性抗体标记、识别、显微操作法富集32例孕周为22～39周的孕妇外周血中的胎儿有核红细胞,引物延伸预扩增后,利用9个短串联重复序列(short tandem repeat,STR)的复合扩增方法对富集的阳性细胞进行扩增,用于孕妇外周血中富集的HbF阳性细胞胎儿来源的遗传学鉴定.结果 经HbF多克隆抗体标记,32名孕妇外周血中均发现与HbF呈阳性反应的胎儿NRBC,并具有鲜明的形态学特征,光学显微镜下可见NRBC细胞质呈棕黄色,核浆比例较低,苏木素复染后胞核呈蓝色,明显区别于其他细胞,每份样本出现NRBC 0.6～1.8个/ml,共计183个,平均为1.3个/ml,经STR多态性基因位点鉴定,准确率为90.6%.结论 利用多肽合成技术制备的抗胎儿血红蛋白γ链的抗体能有效识别母血中的胎儿有核红细胞,可应用于无创性产前基因诊断,具备良好的应用前景.%Objective To investigate the feasibility of a new polyclonal antibody specific to fetal hemoglobin (HbF) and its application in enrichment of circulating fetal nucleated red blood cell(NRBC) for non-invasive prenatal diagnosis. Methods A polyclonal antibody against a synthetic peptide comprising residues 69-78 of the γ-chain of HbF was prepared and conjugated to carrier protein KLH as the immunogen according to the specific antigenic determinant. The peptide-KLH solution was mixed with freund's complete or incomplete adjuvant and immunized goat to prepare specific polyclonal antibody against the γ-chain of fetal hemoglobin. After purification with protein G, maternal blood was obtained from 32 pregnant women at 22 to 39 weeks of gestation. NRBCs were separated and then stained with antibody against the γ chain of HbF. All the positive cells were collected by micromanipulator under microscopic observation, and whole genome was amplified by improved primer extension preamplification (PEP). Multiplex polymerase chain reaction amplification at nine different polymorphic short tandem repeat (STR) loci was also used to determine origin of the positive cells isolated from maternal blood. Results NRBCs stained with antibody against the γ chain of HbF were found in all of the blood from the 32 cases. Attached positive cells with anti-HbF staining have unique morphological characteristics, low nucleus-to-cytoplasm ratio, brown cytoplasm and blue dense nucleus after hematoxylin counterstain under microscopic observation, which can distinguished NRBCs with other cells. A total of 183 NRBCs were found in all of 32 pregnant women at a range of 0.6～1.8 cell/ml venous blood. The accurate rate was 90.6% by the STR genotype identification. Conclusion The antibodies specific to fetal γ-chain of fetal hemoglobin with synthetic peptide technology may have wide clinical utility in identification of fetal NRBCs from maternal circulation for non-invasive prenatal genetic diagnosis.
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