Sharon J, Morrison S L, Kabat E A
Proc Natl Acad Sci U S A. 1979 Mar;76(3):1420-4. doi: 10.1073/pnas.76.3.1420.
A sensitive and rapid method for the detection of monoclonal antibodies secreted by hybridomas is described. Mouse myeloma cells are fused with spleen cells from immunized mice and directly cloned in soft agarose containing selective medium; hybrid clones can be seen after a week. Nitro-cellulose filters that have been coated with a specific protein antigen, with antigen-coupled erythrocyte ghosts, or with other cells used as antigens are then placed on the agarose surface. After incubation to allow immunoadsorption of any secreted antibodies specific for the filter-bound antigen, the filter is removed and overlaid with a suspension of antigen-coupled erythrocytes that react with the adsorbed antibodies; after unbound erythrocytes are allowed to fall off the filter, red spots delineate the sites at which antibody-forming clones are present in the agarose. Alternatively, the filter may be treated with radiolabeled antigen followed by autoradiography. The reliability and sensitivity of the method are demonstrated with alpha-(1 leads to 3)-specific antidextran myeloma J558, and the method's applicability is established by detecting hybridomas with specificities for sheep erythrocytes and for alpha-(1 leads to 3) dextran.
本文描述了一种检测杂交瘤分泌的单克隆抗体的灵敏且快速的方法。将小鼠骨髓瘤细胞与免疫小鼠的脾细胞融合，并直接克隆于含选择性培养基的软琼脂糖中；一周后可见杂交克隆。然后将已包被特定蛋白质抗原、抗原偶联红细胞空壳或用作抗原的其他细胞的硝酸纤维素滤膜置于琼脂糖表面。孵育以使针对滤膜结合抗原的任何分泌抗体进行免疫吸附后，取出滤膜，覆盖与吸附抗体反应的抗原偶联红细胞悬液；未结合的红细胞从滤膜上落下后，红色斑点勾勒出琼脂糖中存在抗体形成克隆的位置。或者，可先用放射性标记抗原处理滤膜，然后进行放射自显影。用α-(1→3)-特异性抗葡聚糖骨髓瘤J558证明了该方法的可靠性和灵敏性，并通过检测对绵羊红细胞和α-(1→3)葡聚糖具有特异性的杂交瘤确定了该方法的适用性。
