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Rationally Designed Ligands for Use in Affinity Chromatography 105
Fig. 5. Schematic representation of the competitive ELISA assay.
no ligand, (iii) protein-HRP and no ligand (corresponding to 100% binding—
inhibition data were calculated relative to this value). For the determination of
the affinity constants, see Note 7.
4. Notes
1. Extent of epoxy activation of agarose beads: Sodium thiosulphate (1.3 M) (3 ml) is
added to 1gofepoxy-activated gel and incubated at room temperature for 20 min.
This mixture is neutralized with 0.1 M HCl and the amount of HCl used registered.
The volume of 0.1 M HCl added corresponds to the number of OH − moles released
(10 μmoles per each 100 μl added), which equals to μmole epoxy groups/g gel.
Therefore, the extent of epoxy activation is expressed as μl HCl used/10 (μmol/g
gel). The protocol usually results in 25-μmol epoxy groups/g moist weight gel.
2. Extent of amination on agarose beads with the TNBS test (16): Aminated gel (0.1
g) is hydrolyzed with 500 μl of 5 M HCl at 50°C for 10 min. Upon cooling,
the hydrolyzed sample is neutralized with 5 M NaOH and added to 1 ml of 0.1
M sodium tetraborate buffer (pH 9.3) and 25 μl of 0.03 M TNBS. Samples are
incubated at room temperature for 30 min prior to measuring their absorbance at
420 nm. The negative control is 1 ml of distilled water to which sodium tetraborate
buffer and TNBS solution (amounts cited above) are added. Calibration curves are
constructed with 6-aminocaproic acid (0–2 μmol/ml). Usual values obtained are
20–25 μmol amine groups/g moist weight gel.
3. Qualitative test for aliphatic amines: A small amount of moist gel (∼1 ml) is placed
on a filter paper and ninhydrin in ethanol (0.2%, (w/v)) sprayed on it. The filter
paper is heated with a hairdryer (very carefully to avoid burning), until development