ANTIGEN-INDEPENDENT BINDING OF RAT IMMUNOGLOBULINS IN A RADIOIMMUNOASSAY - SOLUTIONS TO AN UNUSUAL BACKGROUND PROBLEM
ANTIGEN-INDEPENDENT BINDING OF RAT IMMUNOGLOBULINS IN A RADIOIMMUNOASSAY - SOLUTIONS TO AN UNUSUAL BACKGROUND PROBLEM
复制标题
DOI：
10.1016/0022-1759(84)90243-6
复制
发表时间：
1984-01-01
影响因子：
2.2
通讯作者：
SJOGREN, HO
中科院分区：
文献类型：
--
作者：
NILSSON, R;SJOGREN, HO
当在间接125 I标记的蛋白A测定中检测细胞表面结合的各种类别和IgG亚类的大鼠IG时，注意到大量大鼠血清中IG与塑料表面的非特异性结合。在所有类型的IG中都发现了这种非特异性结合。结合程度随所用试验板的类型而变化，并且在从相同供体连续抽取的血清中随时间波动。用补充有BSA [牛血清白蛋白]、明胶或纤维蛋白原的胎牛血清包被试验威尔斯孔不能消除反应。IG直接与聚苯乙烯结合，而不与胎牛血清中存在的抗原或大鼠血清中的自身抗原结合。使用两种不同的方法来消除非特异性反应。当活细胞用作靶抗原时，仅用非离子去污剂Nonidet P40洗脱细胞结合放射性，其溶解细胞膜而不破坏蛋白A/兔IgG、兔IgG/大鼠IG或大鼠IG/塑料相互作用。当对吸附在非组织培养级平板上的靶抗原检测大鼠血清抗体时，可通过在孵育混合物中加入0.05%吐温20来避免非特异性结合。
A high non-specific binding of Ig to plastic surfaces was noted with a number of rat sera when tested in an indirect 125I-labeled protein A assay for detection of cell-surface-bound rat Ig of various classes and IgG subclasses. This type of non-specific binding was found with all types of Ig. The degree of binding varied with the type of test plate used and fluctuated with time among sera drawn sequentially from the same donors. Coating test wells with fetal calf serum supplemented with BSA [bovine serum albumin], gelatin or fibrinogen did not eliminate the reactions. The Ig bind directly to the polystyrene, and not to antigens present in fetal calf serum or autoantigens in rat serum. Two different approaches were used to eliminate the nonspecific reaction. When living cells were used as target antigens, exclusively cell-bound radioactivity was eluted with the non-ionic detergent Nonidet P40, which solubilizes the cell membrane without breaking the protein A/rabbit IgG, rabbit IgG/rat Ig, or rat Ig/plastic interactions. When rat serum antibodies are tested on target antigens adsorbed on non-tissue culture grade plates, non-specific binding may be avoided by including 0.05% Tween 20 in the incubation mixture.