Huie M A, Cheung M C, Muench M O, Becerril B, Kan Y W, Marks J D
Department of Dermatology, University of California, San Francisco, CA 94143, USA.
Proc Natl Acad Sci U S A. 2001 Feb 27;98(5):2682-7. doi: 10.1073/pnas.051631798.
The ability to isolate fetal nucleated red blood cells (NRBCs) from the maternal circulation makes possible prenatal genetic analysis without the need for diagnostic procedures that are invasive for the fetus. Such isolation requires antibodies specific to fetal NRBCs. To generate a panel of antibodies to antigens present on fetal NRBCs, a new type of nonimmune phage antibody library was generated in which multiple copies of antibody fragments are displayed on each phage. Antibody fragments specific for fetal NRBCs were isolated by extensive predepletion of the phage library on adult RBCs and white blood cells (WBCs) followed by positive selection and amplification on fetal liver erythroid cells. After two rounds of selection, 44% of the antibodies analyzed bound fetal NRBCs, with two-thirds of these showing no binding of WBCs. DNA fingerprint analysis revealed the presence of at least 16 unique antibodies. Antibody specificity was confirmed by flow cytometry, immunohistochemistry, and immunofluorescence of total fetal liver and adult RBCs and WBCs. Antibody profiling suggested the generation of antibodies to previously unknown fetal RBC antigens. We conclude that multivalent display of antibodies on phage leads to efficient selection of panels of specific antibodies to cell surface antigens. The antibodies generated to fetal RBC antigens may have clinical utility for isolating fetal NRBCs from maternal circulation for noninvasive prenatal genetic diagnosis. Some of the antibodies may also have possible therapeutic utility for erythroleukemia.
从母体循环中分离胎儿有核红细胞（NRBCs）的能力使得无需对胎儿进行侵入性诊断程序即可进行产前基因分析成为可能。这种分离需要针对胎儿NRBCs的特异性抗体。为了产生一组针对胎儿NRBCs上存在的抗原的抗体，构建了一种新型的非免疫噬菌体抗体文库，其中每个噬菌体上展示多个抗体片段拷贝。通过在成人红细胞和白细胞（WBCs）上对噬菌体文库进行广泛的预清除，然后在胎儿肝脏红系细胞上进行阳性选择和扩增，分离出对胎儿NRBCs特异的抗体片段。经过两轮选择，所分析的抗体中有44%与胎儿NRBCs结合，其中三分之二不与WBCs结合。DNA指纹分析显示存在至少16种独特的抗体。通过对整个胎儿肝脏以及成人红细胞和白细胞进行流式细胞术、免疫组织化学和免疫荧光分析，证实了抗体的特异性。抗体谱分析表明产生了针对先前未知的胎儿红细胞抗原的抗体。我们得出结论，噬菌体上抗体的多价展示导致高效选择针对细胞表面抗原的特异性抗体组。所产生的针对胎儿红细胞抗原的抗体可能在从母体循环中分离胎儿NRBCs以进行非侵入性产前基因诊断方面具有临床应用价值。其中一些抗体也可能在治疗红白血病方面具有潜在的治疗用途。
