PRELIMINARY EVALUATION OF THE ASTED-XL DIALYSIS SYSTEM TOWARDS ITS APPLICABILITY IN LIGAND-BINDING ASSAYS

In ligand binding assays, the separation of bound and free fraction of the labeled ligand is very important. Dialysis is generally overlooke d as separation technique since it requires large volumes and long ana lysis times. The availability of the ASTED-system (Automated Sequentia l Trace Enrichment of Dialysates) might open ways for a complete autom ation of immune assays including the separation step. A well-documente d radio-immune assay for 3-keto-desogestrel (Org3236) was used to test the potentials of this system. The tritiated analog of Org3236 not on ly served as label in the immune assay but was also used to trace this compound in the entire procedure. The dialysis efficiency increased w ith the dialysis time and with the flush rate of the recipient solvent (tris-HCl or phosphate buffer). Addition of methanol to recipient sol vent had spectacular effects on the recovery. With tris-HCl buffer, 0. 18 mL/min and 1.0 mL recipient solvent 2.5% of the label was collected . Addition of 50% methanol resulted in a 5-fold increase to 12%. Repla cement of buffer by 100% methanol resulted in another 5% increase in d ialysis efficiency which was accompanied by a reduction in the antibod y binding in the donor compartment due to denaturation of the antibody . The commercial availability of other types of membranes is essential to find optimal conditions for each analyte. A serious problem is the carry-over effect between subsequent samples. Roughly 0.25% of label was collected in the next run which may have a substantial impact on t he accuracy and precision of the assay. Renewal of the dialysis membra ne might exclude this carry-over effect but is not a serious option wi th the available instrumentation. Automated dialysis systems can be ve ry valuable for ligand binding assays as soon as membranes become avai lable for the analytes of interest which provide high recoveries (>40% ) in 1 mL recipient solvent. Moreover their carry-over effect should b e negligible or eliminated by more efficient rinsing procedures of the entire dialysis system. Temperature control is favourable for the imm une assays as well as for the dialysis process in that the kinetics ar e temperature dependent.