弱抗原血型快速且準確篩檢法之開發

Abstract

有鑒於血型 RhD 型之中的 Del 血型(D elution)無法快速的藉由一般捐血中心的血型篩檢的方式(Ortho BioVue System Microtyping card)來判別為 RhD 陽性反應，造成日後所產生的輸血上之疾病，如新生兒溶血症(hemolytic disease of newborn, HDN)。而傳統篩檢 Del 血型的方法，吸附沖出法過於費時(1小時)、須過多檢體(1c.c.紅血球)與花費過高(1c.c.試劑)，無法應用於輸血前試驗(pretansfusion testing)。針對此問題，我們設計了在血液中增強血型抗原表現弱化的紅血球凝集的方法，此設計概念來自於血型抗原數稀少，使得無法具有足夠的血型抗體反應促使紅血球凝集。因此，只要有足夠的血型抗原仍然有機會造成紅血球凝集。基於此概念，我們嘗試了四種不同的放大技術：第一種是利用一水溶性高分子 poly(acrylic acid) ，修飾上抗 anti-(RhD) antibody 的抗體(anti-mouse IgG)，進而使得弱血型之血球可以藉由高分子上之二級抗體來達到凝集之效果；第二種是利用一水溶性高分子poly(allylamine)，先將其上之 amine group 部份乙醯化(acetylation)，來減少和血球細胞的非特異性鍵結(non-specific binding)之後再修飾上biotin形成新的高分子材料(Ac-pAAm-biotin)，藉由 anti-(RhD) antibody、 anti-(mouse IgG), biotin conjugates、 Ac-pAAm-biotin、avidin 四者交互作用來達到弱血型凝集目的；第三種和第四種是將新的高分子材料(Ac-pAAm-biotin)，置換成現有的兩種 biotin 修飾過的商品， bovine serum albumin, biotin conjugates和poly(acrylic acid) beads, biotin conjugates 來比較弱血型血液凝集的效果。由我們的實驗結果顯示，只有 bovine serum albumin, biotin conjugates 這組方式才有辦法達到快速(10分鐘)、檢體少(10λ，3%紅血球)、試劑少(20λ試劑)、準確性高、再現性高的標準，符合血庫快速篩檢之目的。

There is a problem that the false negatives of red blood cells(RBCs) with Rh(Del), causing hemolytic disease during blood transfusion and hemolytic disease of newborn (HDN), is sometimes happened in our blood centers where medical technologists identify them in general blood typing cards (Ortho BioVue System Microtyping card). Adsorption and elution method, the standard procedure of identifying Del, spends more times (at least an hour), quantities of sample (1c.c. RBCs), and costs (1c.c. reagent) than the method of microtyping card so that they do not use this method at pretransfusion testing. To solve this problem, we suppose that the appearance of false negative is due to less D antigens per RBC membrane than D positive persons. RBCs have no probability to agglutinate by monoclonal anti-(RhD) antibody. If we amplify D antigens from RBCs with Del, they could have been agglutinated because of the probability of agglutination arising. Base on this conception, we test four different kinds of antigen-antibody amplification techniques: the first is that a water soluble polymer, poly(acrylic acid), conjugated with secondary antibody, anti-(mouse IgG) which binds on anti-(RhD) antibody, would be used as an cross-linker that let RBCs with Del agglutinate; the second is that a new polymer (Ac-pAAm -Biotin), made from a water soluble polymer, poly(allylamine) which was partially acetylation and biotinylation, would be interact with anti-(RhD), anti-(mouse IgG)-biotin conjugates, and avidin to agglutinate RBCs with Del. The third and fourth are that alternatives of the new polymer (pAAm-Ac-Biotin), commercial bovine serum albumin, biotin conjugates (BSA-biotin) and poly(acrylic acid) beads, biotin conjugates (pAA beads-biotin), would be applied to agglutination of RBCs with Del. Our result revealed that only the third method, BSA-biotin, can prevent false negative efficaciously and this method is more rapid (about 10 mins) and less quantities of sample (10λ, 3% RBCs) and costs (20λ reagent) than adsorption and elution method.