Kinetic aspects of the antigen-antibody reaction in various radioimmunoassays: effect of delayed addition of labeled or unlabeled antigens on sensitivity of assay.
Kinetic aspects of the antigen-antibody reaction in various radioimmunoassays: effect of delayed addition of labeled or unlabeled antigens on sensitivity of assay.
复制标题
各种放射免疫测定中抗原抗体反应的动力学方面：延迟添加标记或未标记抗原对测定灵敏度的影响。
DOI：
10.1016/0009-8981(79)90169-4
复制
发表时间：
1979
期刊：
影响因子：
--
通讯作者：
K. Miyai
中科院分区：
文献类型：
--
作者：
K. Ichihara;Toshihide Yamamoto;M. Azukizawa;K. Miyai
本文用四种双抗体放射免疫分析法（RIA）系统地研究了抗原抗体反应动力学。在所有RIA中，延迟加入标记抗原24 - 48 h后获得的剂量-反应曲线（曲线B）与同时加入试剂获得的剂量-反应曲线（曲线A）相比向下和向左移动，从而提高了测定的灵敏度。相反，延迟加入未标记抗原获得的剂量-反应曲线（曲线C）向上移动并移到曲线A的右侧，导致灵敏度降低。在人促甲状腺激素（hTSH）RIA中，曲线B和C即使在孵育168 h后也很小地接近曲线A。用5种不同来源的抗hTSH抗血清在两种孵育温度下观察到类似的现象，125 I标记的hTSH和未标记的hTSH的稀释曲线似乎是平行的。因此，hTSH RIA观察到的现象不能归因于测定条件或所用试剂的特殊性质。在胰岛素RIA中，曲线B和C的移动的逆转是轻微的，但与在hTSH RIA中观察到的那些相似。在1- 3，5，3 '-三碘甲状腺原氨酸放免试验中，曲线B和C随孵育时间延长逐渐接近曲线A，孵育98 h后曲线B与曲线A基本一致。另一方面，在甲胎蛋白（AFP）RIA中，曲线B和C不接近曲线A，即使延长孵育时间至288 h。抗体的“平衡亲和常数”具有相同的数量级，因此常数的差异不太可能解释这些RIA可逆性的差异。在APF RIA中，在3000 ×g离心后，无第二抗体沉淀出大量的抗原-抗体复合物。这些发现表明，免疫复合物的解离程度取决于它们的大小，这反过来又与抗原的分子量有关。
The kinetics of the antigen-antibody reaction were examined systematically in four kinds of double-antibody radioimmunoassay (RIA). In all the RIAs, the dose-response curves obtained on delayed addition by 24 to 48 h of labeled antigens (curves B), were shifted downwards and to the left of those obtained on simultaneous addition of the reagents (curves A), resulting in improved sensitivity of the assay. On the contrary, the dose-response curves obtained on delayed addition of unlabeled antigens (curves C), were shifted upwards and to the right of curves A, resulting in reduced sensitivity. In human thyrotropin (hTSH) RIA, curves B and C approached curves A very little, even after 168 h of incubation. A similar phenomenon was observed with anti-hTSH antisera from five different sources at two incubation temperatures, and the dilution curves of125I-labeled hTSH and unlabeled hTSH appeared to be parallel. Therefore, the phenomenon observed with hTSH RIA could not be attributed to the assay conditions or to peculiar properties of the reagents used. In insulin RIA, the reversibilities of the shifts of curves B and C were slight but comparable to those observed in hTSH RIA. In 1–3,5,3'-triiodothyronine RIA, curves B and C gradually approached curves A on prolonged incubation and curves B became nearly identical with curves A after 98 h of incubation. On the other hand, in α-fetoprotein (AFP) RIA, curves B and C did not approach curves A, even on prolonged incubation for up to 288 h. The “equilibrium affinity constants” of the antibodies were of the same order of magnitude, thus it is unlikely that differences in the constants can account for the differences in the reversibility of these RIAs. In APF RIA, a significant amount of the antigen-antibody complex was precipitated without second antibody after centrifugation at 3000 ×g. These findings suggest that the extent of dissociation of the immune complexes depends on their size, which in turn is related to the molecular weight of the antigen.