| Abstract: | Abstract In ligand binding assays, the separation of bound and free fraction of the labeled ligand is very important. Dialysis is generally overlooked as separation technique since it requires large volumes and long analysis times. The availability of the ASTED-system (Automated Sequential Trace Enrichment of Dialysates) might open ways for a complete automation of immuno assays including the separation step. A well-documented radio-immuno assay for 3-keto-desogestrel (Org3236) was used to test the potentials of this system. The tritiated analog of Org3236 not only served as label in the immuno assay but was also used to trace this compound in the entire procedure. The dialysis efficiency increased with the dialysis time and with the flush rate of the recipient solvent (tris-HCI or phosphate buffer). Addition of methanol to recipient solvent had spectacular effects on the recovery. With tris-HCI 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%. Replacement of buffer by 100% methanol resulted in another 5% increase in dialysis efficiency which was accompanied by a reduction in the antibody 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 carryover effect between subsequent samples. Roughly 0.25% of label was collected in the next run which may have a substantial impact on the accuracy and precision of the assay. Renewal of the dialysis membrane might exclude this carry-over effect but is not a serious option with the available instrumentation. Automated dialysis systems can be very valuable for ligand binding assays as soon as membranes become available for the analytes of interest which provide high recoveries (>40%) in 1 mL recipient solvent. Moreover their carry-over effect should be negligible or eliminated by more efficient rinsing procedures of the entire dialysis system. Temperature control is favourable for the immuno assays as well as for the dialysis process in that the kinetics are temperature dependent. |
