CAJ | 학술논문

目的筛选血型A抗原表位的模拟多肽,探讨其在血型A抗体检测中的应用价值.方法用血型A抗原特异性单克隆抗体NaM87-1F6从噬菌体随机12肽库中筛选能与之结合的噬菌体展示肽.采用噬菌体ELISA、噬菌体微筛选试验鉴定噬菌体与抗体的结合力.DNA测定并推断特异性噬菌俸克隆展示的多肽序列.凝集抑制试验检测单克隆噬菌体对抗体凝集红细胞的抑制作用,ABO-ELISA初步分析噬菌体展示多肽检测血型抗体的效能.结果经筛选和鉴定后得到7个阳性克隆,其中6个展示多肽序列为EYWYCGMNRTGC(C5),1个为QIWYERTLPFTF(C17).凝集抑制试验提示2个噬菌体展示多肽可以特异性抑制NaM87-1F6对A型红细胞的凝集作用.基于噬菌体多肽C5、C17的ABO-ELISA检测血浆抗A抗体试验的受试者操作特性曲线下面积分别为0.889(P=0.000)和O.751(P=0.000),ABO-ELISA值与凝集试验抗体滴度间的Spearman相关系数分别为0.743(P＜0.01)和0.664(P＜0.01).基于C5的ABO-ELISA在临界值为0.300时其敏感度、特异度分别为82.2%和83.3%;基于C17的ABO-ELISA在临界值为0.250时其敏感度、特异度分别为68.9%和63.3%.结论多肽EYWYCGMNRTGC,QIWYERTLPFTF可以模拟血型A抗原的表位.基于这些多肽的ABO-ELISA方法具有检测血型A抗原特异性抗体的潜能.
목적 사선혈형A항원표위적모의다태,탐토기재혈형A항체검측중적응용개치.방법 용혈형A항원특이성단극륭항체NaM87-1F6종서균체수궤12태고중사선능여지결합적서균체전시태.채용서균체ELISA、서균체미사선시험감정서균체여항체적결합력.DNA측정병추단특이성서균봉극륭전시적다태서렬.응집억제시험검측단극륭서균체대항체응집홍세포적억제작용,ABO-ELISA초보분석서균체전시다태검측혈형항체적효능.결과 경사선화감정후득도7개양성극륭,기중6개전시다태서렬위EYWYCGMNRTGC(C5),1개위QIWYERTLPFTF(C17).응집억제시험제시2개서균체전시다태가이특이성억제NaM87-1F6대A형홍세포적응집작용.기우서균체다태C5、C17적ABO-ELISA검측혈장항A항체시험적수시자조작특성곡선하면적분별위0.889(P=0.000)화O.751(P=0.000),ABO-ELISA치여응집시험항체적도간적Spearman상관계수분별위0.743(P＜0.01)화0.664(P＜0.01).기우C5적ABO-ELISA재림계치위0.300시기민감도、특이도분별위82.2%화83.3%;기우C17적ABO-ELISA재림계치위0.250시기민감도、특이도분별위68.9%화63.3%.결론 다태EYWYCGMNRTGC,QIWYERTLPFTF가이모의혈형A항원적표위.기우저사다태적ABO-ELISA방법구유검측혈형A항원특이성항체적잠능.
Objective To explore the peptides that can mimic the blood type A antigen and evaluate the anti-A antibody detection value of these peptides.Methods The anti-A monoclonal antibodv (NaM87-1F6)was used to panning the phage clones from a phage display 12-mer peptide library.Positive clones were identified by phage ELISA,phage mieropanning methods.Phage DNA Was sequenced and the corresponding peptide sequences were deduced.Agglutination inhibition test WaS performed to assess the ability of phage clones to inhibit the binding between the type A red blood cell and the anti-A antibody. ABO-ELISA based on the selected peptides was compared with classical haemagglutination test jn the detection of senlm anti-A antibody.Results Seven positive clones were chosen after panning,phage ELISA and phage micropanning.Six clones displayed peptide EYWYCGMNRTGC(C5),the other one displayed peptide QIWYERTLPFTF(C17).The phages displaying the selected peptides could specifically inhibit agglutination of type A red blood cells(RBCs)by anti-A antibodies.In the ABO-ELISA based on C5 and C17,the receiver operating characteristic(ROC)Curve showed that area under curve(AUC)were 0.889 (P=0.000),0.75l(P=0.000)respectively.The Spearman correlation Coeffieient between the ABO-EliSA value and the antibody titer derived from haemagglutination assay were 0.743(P<0.01),0.664(P<0.01)respectively.As for C5,0.300 was the best cut-off for ABO-ELISA with 82.2% sensitivity and 83.3% specificity.As for C17,the sensitivity and specificity of ABO-ELISA was 68.9% and 63.3% respectively when the cut-off value was 0.250.Conclusions The peptides EYWYCGMNRTGC and QIWYERTLPFTF can mimic the blood type A antigenic epitope.ABO-ELISA based on these peptides has the potential for the detection of anti-A antibody.