Characterization of the Asialofetuin microtitre plate-binding assay for evaluating inhibitors of ricin lectin activity.
Optimum conditions for the binding of ricin to the glycoprotein asialofetuin immobilized on microtitre plates were investigated for the purpose of evaluating inhibitors of ricin B-chain lectin activity. Such inhibitors are of potential value in the use of immunotoxins based on ricin. This assay was first reported in 1986, but has not been characterized fully. Maximum binding of asialofetuin to the plate was observed at a concentration of ca. 4 microg ml(-1). Binding increased with time of incubation (1-24 h), pH (7.4-9.9) and temperature (2-37 degrees C). The pH effects were more marked at lower temperatures. Saturable binding of ricin to immobilized asialofetuin was observed, and at least 80% of maximum binding was observed by 10 min of incubation time. The binding was found to be very tight, such that an appreciable proportion of ricin added to the wells was bound at low concentrations, and binding was only partially reversible by addition of free galactose. Consequently, only estimates of the ricin-asialofetuin and ricin-galactose dissociation constants could be determined: 1.9 nM and 83 microM, respectively. Binding of ricin A- and B-chains was found to be 47% (at a 200-fold higher concentration) and 26% (at a twofold higher concentration) of that of the whole ricin molecule, respectively. The assay permits qualitative comparison of inhibitors of ricin B-chain lectin activity.
Dawson
R M
RM
Aeronautical and Maritime Research Laboratory, Defence Science and Technology Organization, Melbourne, Victoria, Australia. ray.dawson@dsto.defence.gov.au
Paddle
B M
BM
Alderton
M R
MR
D001212:Asialoglycoproteins / Q000737:chemistry / Q000378:metabolism*; D001667:Binding, Competitive / Q000187:drug effects; D002021:Buffers; D004305:Dose-Response Relationship, Drug; D060748:Fetuins; D005690:Galactose / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D008828:Microbiological Techniques / Q000295:instrumentation; D011485:Protein Binding / Q000187:drug effects; D012276:Ricin / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors; D000509:alpha-Fetoproteins / Q000737:chemistry / Q000378:metabolism*
D001212:Asialoglycoproteins; D002021:Buffers; D060748:Fetuins; D000509:alpha-Fetoproteins; C017029:asialofetuin; D012276:Ricin; D005690:Galactose