双向免疫扩散法(doubleradialimmunodiffusion).ppt

三.抗球蛋白媒介凝集分析(Antiglobulin-mediated agglutination) 利用抗人體免疫球蛋白作為二次抗體，使結合在紅血球表面的Anti-IgG不完全抗體，完成凝集反應，這種體驗方法就稱為Coombs’ test。 Direct Coombs’ test：適用於測定在體內已被致敏化的紅血球，以證明體內的紅血球附著有不完全抗體。 Indirect Coombs’ test：可用來測定患者血清中所含的不完全抗體。 Rose-Waaler test：先從兔子提引出抗綿羊紅血球抗體IgG ，然後將抗體濃度稀釋到不會使綿羊回紅血球產生凝集反應，紅血球使之致敏化，作為檢驗試劑，測患者血清中是否含有類風濕性因子。 Direct Coombs’ test 利用抗人類免疫球蛋白作為二抗使結合在RBC表面的anti-Rh IgG不完全Ab完成凝集 常用來檢查自體抗體引發的溶血疾病或血型不合的輸血反應 zeta potential Direct Coombs’ test: 適於測定體內已被致敏化的RBC, 以證明RBC附著有不完全Ab Indirect Coombs’ test: 可測定serum所含的不完全Ab Rose-waaler test從兔子提引出抗綿羊RBC IgG,將Ab稀釋到不會使綿羊RBC產生凝集,加入綿羊RBC使之致敏化若患者有類風濕因子(RF),將能與致敏化RBC表面IgG結合,形成凝集反應 Agglutination inhibition 游離Ag(待測檢體)先與有限的抗Ab反應，然後加入Ag sensitized 粒子，游離Ag與粒子表面的Ag相同，會共同競爭抗Ab。 Enhancement media Media Mode of action Albumin Changes Charge for phase 2 (lattice formation) enhancement Low ionic strength solution Increase antibody uptake in phase 1 (Antibody binding to antigen) Polyethylene glycol Increase antibody uptake in phase 1 Proteolytic enzyme Decease the distance between cells by decreasing the zeta potential; phase 2 is enhanced Polybrene Decease the distance between cells by decreasing the zeta potential; phase 2 is enhanced Agglutination Precipitation Insoluble or particulate antigen or antibody soluble antigen or antibody Antigen must have at least two antigenic determinants Antigen must have at least two antigenic determinants Antigen excess results in a postzone reaction Antigen excess results in a postzone reaction Antibody excess results in a prozone reaction Antibody excess results in a prozone reaction Reaction time:minutes to hours Reaction time:hours to days Test result:quantitative or semiquantitative Test result:quantitative, semiquantitative, or qualitative An animal is immunized with bovine serum albumin to which 2,4-dinitrophenol has been conjugated (DNP-BSA). Immune serum is obtained and placed in the center well of an Ouchterlony plate, with antigens in the outer wells as shown below. If BSA share sequen