抗紫红笛鲷血清免疫球蛋白单克隆抗体的制备

PREPARATION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST SERUM IMMUNOGLOBULINS OF LUTJANUS ARGENTIMACULATUS

采用蛋白A sephrose亲和层析法,分离提纯了紫红笛鲷Lutjanusargentimaculatus的血清免疫球蛋白(Ig),并制备了抗紫红笛鲷血清Ig分子的单克隆抗体。经间接ELISA、免疫印迹(westernblotting)的检测和鉴定,得到抗紫红笛鲷Ig分子重链的单抗6株,分别为IgG11株、IgG23株、IgG2a1株、IgG31株。这6株抗紫红笛鲷Ig的单抗各有1株与同为鲈形目的青石斑鱼Epinephelusawoara和尖吻鲈Latescalcarifer的Ig分子有很弱的交叉反应,而同鲽形目的牙鲆Paralichthysolivaceus的Ig分子没有交叉反应,具有较强的种间特异性;同时揭示,同目不同属的鱼类血清Ig之间的相似度较低,不同目之间的相似度极低。

The serum immunoglobulins (Igs) of mangrove red snapper Lutjanus argentimaculatus(Forskl),purified by affinity chromatography on protein Asepharose,were used as antigens in preparation of monoclonal antibodies(McAbs).After selected by HAT medium and enzymelinked immunosorbent assay(ELISA),and cloned,six McAbs against serum Igs of L.argentimaculatus were produced and characterized.All of the McAbs are functional for use in ELISAs and immunoblotting applications.Characterizations of the McAbs for mouse Ig class and subclass indicated that among the six McAbs,one was IgG1,three were IgG2,one was IgG2a,and one was IgG3.Western blot analysis of the six hybridomas with purified serum Ig of L.argentimaculatus indicated that all the six McAbs were heavy chain specific.ELISAs indicated that one of the six McAbs had weak reaction with serum Igs of banded grouper WTEpinephelus awoara,another one had a weak reaction with those of white seabass Lates calcarife,and no McAb reacted with those of Japanese flouder Paralichthys olivaceus.The results showed that the fish McAbs had strong species specificity,whereas the similarity of serum Igs among different genera was low and it was very low among different orders.