Z. Huang et al., PRELIMINARY EVALUATION OF THE ASTED-XL DIALYSIS SYSTEM TOWARDS ITS APPLICABILITY IN LIGAND-BINDING ASSAYS, Analytical letters, 28(15), 1995, pp. 2635-2651
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.