Development and assessment of radioreceptor binding assays for the detection of saxitoxin binding proteins in biological extracts.
Several radioreceptor assays using tritiated saxitoxin ([(3)H]STX) were developed to identify a suitable primary screening method for the detection and characterization of soluble saxitoxin binding proteins from biological extracts. Assays using anion and cation exchange, protein binding, and traditional charcoal radioreceptor methods were compared with two previously reported formats. A protein binding assay incorporating filters of mixed cellulose esters (MCE) outperformed all other assay strategies with maximal signal, low background, exceptional reproducibility, minimal matrix effects, and high throughput. Binding site titrations verified that an increase in total protein in the assay led to a concomitant linear increase in the amount of specifically bound [(3)H]STX within the range of 1-90microg total protein. Saturation binding experiments demonstrated that the binding sites were saturable and that nonspecific binding was linear. The MCE assay was unaffected by 600mM NaCl and 500mM KCl. Likewise, minimal variation of specific binding was observed between pH 5 and pH 9, but inhibition was observed below pH 5.
Robertson
Alison
A
Department of Biochemistry and Molecular Biology, James Cook University, Townsville, Queensland 4811, Australia. alison.robertson@nrc-cnrc.gc.ca
Negri
Andrew P
AP
Burnell
James N
JN
Llewellyn
Lyndon E
LE
D029845:Amphibian Proteins / Q000032:analysis / Q000378:metabolism; D000818:Animals; D001181:Arthropods / Q000378:metabolism; D002352:Carrier Proteins / Q000032:analysis* / Q000378:metabolism; D002482:Cellulose; D005374:Filtration; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007474:Ion Exchange; D011485:Protein Binding; D011869:Radioligand Assay / Q000379:methods*; D012492:Salts; D012530:Saxitoxin / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000032:analysis / Q000378:metabolism
D029845:Amphibian Proteins; D002352:Carrier Proteins; D012492:Salts; D029961:Zebrafish Proteins; D012530:Saxitoxin; D002482:Cellulose