Schliebs R, Rothe T, Bigl V
Biomed Biochim Acta. 1983;42(5):537-46.
The efficiency of separation of the radioligand receptor complex from the unbound ligand by both rapid filtration and centrifugation is compared for two standard receptor assays in the rat brain: the beta-adrenergic receptor using 3H-dihydroalprenolol and the benzodiazepine receptor using 3H-flunitrazepam. Whereas in beta-adrenergic binding studies both separation procedures yield similar binding data, the apparent binding in the benzodiazepine receptor assay is lower in the filtration experiments as compared to the data obtained by centrifugation, indicating an incomplete retention of the ligand receptor complex by filter. However, in both assays the results of the binding studies are highly dependent on the type of filters used. The following filters were compared: glass fibre filters GE/B and GF/C (Whatman Ltd., Kent, England), membrane filter HAWP (pore size 0.45 micrometer, Millipore Corporation, Bedford, U.S.A.) and glass fibre filters NK I, NK VIII, NK-15-90, NK 13-90, NK 14-120 (all produced by VEB Feinpapierfabrik Neu-Kaliss, G.D.R.). Although a somewhat higher specific binding can be obtained by the membrane filter and several types of the NK filters as compared to the most widely used Whatman GF/C the manifold higher unspecific binding of the radioligand by these filters does not favour their use in ligand assays. Retention as well as unspecific binding of the filter NK VIII is very similar to the Whatman GF/C filter. This new filter seems, therefore, well suited for radioligand studies.
在大鼠脑的两种标准受体测定中,比较了通过快速过滤和离心将放射性配体-受体复合物与未结合的配体分离的效率:使用³H-二氢阿普洛尔的β-肾上腺素能受体测定和使用³H-氟硝西泮的苯二氮䓬受体测定。在β-肾上腺素能结合研究中,两种分离方法产生的结合数据相似,而在苯二氮䓬受体测定中,过滤实验中的表观结合低于离心获得的数据,这表明过滤器对配体-受体复合物的保留不完全。然而,在这两种测定中,结合研究的结果高度依赖于所使用的过滤器类型。比较了以下过滤器:玻璃纤维过滤器GE/B和GF/C(英国肯特郡的Whatman有限公司)、膜过滤器HAWP(孔径0.45微米,美国贝德福德的密理博公司)以及玻璃纤维过滤器NK I、NK VIII、NK-15-90、NK 13-90、NK 14-120(均由民主德国的VEB Feinpapierfabrik Neu-Kaliss生产)。尽管与最广泛使用的Whatman GF/C相比,膜过滤器和几种类型的NK过滤器可以获得略高的特异性结合,但这些过滤器对放射性配体的非特异性结合要高得多,这不利于它们在配体测定中的使用。过滤器NK VIII的保留以及非特异性结合与Whatman GF/C过滤器非常相似。因此,这种新型过滤器似乎非常适合放射性配体研究。