抗胆汁螺杆菌单克隆抗体的制备

Preparation of Monoclonal Antibodies against Helicobacter bilis

目的制备抗胆汁螺杆菌单克隆抗体(McAbs)。方法用胆汁螺杆菌B2m株皮下免疫BALB/c小鼠,采用杂交瘤技术进行融合。以酶联免疫吸附实验(ELISA)筛选抗胆汁螺杆菌单克隆杂交瘤细胞株并初步鉴定其特异性;免疫印迹试验测定单抗所结合的抗原表位;免疫双向扩散试验确定IgG亚类;腹腔接种法、辛酸-硫酸铵盐析法大量制备、纯化单克隆抗体。结果经过ELISA筛选获得11个阳性杂交瘤细胞株,其效价最高达1:4×105以上,并与实验动物常见的15种病原菌呈阴性反应;IgG亚类为IgG2a和IgG2b;免疫印迹试验显示,6株(A^F)与胆汁螺杆菌大约相对分子质量(172、0、21、30、52、66)×103的抗原特异结合,5株(G^K)皆与胆汁螺杆菌、幽门螺杆菌等三种螺杆菌大约相对分子质量(52、82)×103的抗原呈阳性反应,表明A^F株针对的是胆汁螺杆菌特异性抗原,G^K株可能具有属特异性。结论筛选的单克隆抗体具有较高的特异性和敏感性,所结合的抗原为胆汁螺杆菌或螺杆菌的免疫优势抗原,为进一步的种、属生物学特性研究、菌株分型及血清学检测方法建立等奠定了基础。

Objective To prepare monoclonal antibodies（McAbs） against H. bilis. Method BALB/c mice were immunized with B2m H. bilis hypodermically and McAbs were gained by hybridoma techniques. Hybridoma cells were screened and their specificity was elementarily identified by enzyme-linked immunoabsorbent assay （ELISA） ; Epitopes binding McAbs were detected by Western blotting; Immunodiffusion method was used for detecting IgG subclass; McAbs were mass-produced by celiac inoculation and purificated by caprylic acid-ammonium sulfate fractionation. Result After ELISA, 11 positive hybridoma subcell lines were obtained that didn＇t cross-react with 15 kinds of common pathogenic bacterial strains on laboratory animals and the highest value was greater than 1：4 ×10^5 ; The IgG belonged to IgG2a and IgG2b subclasses; Western blotting results indicated that immunodominant antigens in H. bilis binding 6 McAbs （A- F） were located at about molecular weight （17,20,21,30,52,66） × 10^3 respectively via WB while 5 McAbs （G - K） all at about （52,82） × 10^3 , suggesting that McAbs A - F aimed at specific antigen H. bilis and McAbs G - K probably possessed of genus specificity. Conclusion The 11 McAbs had high specificity and sensitivity and antigens binding them were immunodominant ones for H. bilis or Helicobacter spp., which could be used in the further study including biological characteristics of species and genus, strain typing and sera detecting.