br Immune affinity subtraction of transferrin br Immune
2.5. Immune affinity subtraction of transferrin
Immune affinity subtraction of transferrin was carried out using AffiAmino Ultrarapid Agarose beads (Lab on a Bead) according to manufacturer's specifications. The first step includes the activation of the beads surface for the iodinated anti-transferrin antibody coupling. For this, 10 µL of the bead suspension (10% bead suspension in 15 mM phosphate pH 7.4, 150 mM NaCl, 20% ethanol) was placed in a 2 mL eppendorf to remove the storage solution by capturing the beads at the bottom of the vial with the help of a magnet. Afterwards, the beads were re-suspended in 100 µL of the binding buffer (PBS) and gently
mixed. The buffer was removed by magnetic separation and this pro-cedure was repeated three times. The next step was the covalent cou-pling of the iodinated anti-transferrin antibody to the amino-reactive groups on the activated beads (10 µL of 10% suspended beads) by mixing these beads with 100 µL of the iodinated antibody solution (50 µg mL−1 in the binding buffer, PBS) for 1 h on gently stirring at room temperature. Afterwards, the solution, which contains the excess of antibody, was removed by magnetic separation and the beads were washed three times with 100 µL of PBS, then the remaining amino-re-active groups on the beads were blocked with 8 µL of ethanolamine (50% in binding buffer, PBS) by gently stirring for 45 min at room temperature. Finally, the beads were washed twice with 100 µL of the binding buffer (PBS) and re-suspended in 100 µL of the same buffer. This bead suspension was used for immune subtraction of transferrin. For this, the liquid was removed by magnetic separation and then the beads, which are coated with the iodinated-antitransferrin antibody, were incubated with 100 µL of the transferrin solution (10 and 20 µg mL−1 in the binding buffer, PBS) for 1 h at room temperature on gently stirring. Then the non-bound fraction was removed and collected by magnetic separation and the beads were washed twice with 100 µL of the binding buffer (PBS).
2.6. Sandwich immunoassay coupled to ICP-MS detection
Aliquots of 100 µL of H2DCFDA lysate samples or transferrin standard solutions (10 and 15 µg mL- in the binding buffer, PBS) were mixed with 20 µL of the biotinylated anti-transferrin antibody (1 mg mL−1 in the binding buffer, PBS) and 20 µL of the iodinated anti-transferrin anti-body (1 mg mL−1 in the binding buffer, PBS) in an Eppendorf tube and incubated for 15 min at room temperature to form a sandwich immune complex. Then, 350 µL of the streptavidin-coated magnetic micro-particles suspension was added and incubated for another 15 min to allow the capture of the immune complex due to streptavidin-biotin interactions. Subsequently, the magnetic microparticles with the formed immune complex were separated from the solution under the external magnetic field provided by a magnet. The liquid was discarded and the microparticles attached to the bottom of the vial were washed three times with PBS buffer, twice with Milli Q water, and then re-suspended in 100 µL of 0.1% HNO3 (Suprapur). An aliquot of 20 µL of this solution was directly injected into the flow injection system (FI-DF-ICP-MS) for monitoring of iodine. All of the measuring results are the average results for triplicate analysis unless otherwise stated.
3. Results and discussion
3.1. Iodination using iodination beads
The iodination of peptides and proteins (e.g antibodies) is a common strategy by using the longer half-life radioactive isotope 125I. The chemistry of the reaction has been widely studied and it involves incorporation of 125I into tyrosine or histidine residues within the peptide or protein through electrophilic substitution for hydrogen causing minimal alteration to the protein backbone. The reaction is done by treating the sample with Na125I in the presence of enzymatic or chemical oxidants to generate a source of electrophilic iodine. Here, we have tried to use the advantage of the commercial iodination systems on a solid surface (Thermo Scientific™ Pierce™ iodination beads) con-sisting of non-porous, polystyrene beads of 3 mm-diameter coated with an oxidizing reagent, which provides efficient activation of iodine for protein iodination using natural NaI. The mild nature of the iodination reaction using this system permits to maintain the biological activity of proteins than using soluble oxidants. Moreover, the iodination reaction is terminated once the beads are removed from the solution, which simplifies the labelling protocol substantially as described by other authors [27,28].
Thus, the anti-transferrin antibody was iodinated using the
Fig. 2. Chromatograms obtained for the iodinated anti-transferrin antibody using SEC-ICP-MS monitoring: (A) 127I and (B) 32S.