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  • br previously described protocol and the success of the


    previously described protocol and the success of the iodination reaction was monitored by using SEC-ICP-MS. The chromatogram corresponding to the iodinated antibody is shown in Fig. 2. The iodine trace is shown in Fig. 2A and the sulfur trace in Fig. 2B (acquired in low and medium resolution respectively by DF-ICP-MS). As can be observed, the iodi-nation reaction seems to produce a relatively clean product without detectable degradation of the antibody. The stoichiometry of the iodi-nated species was obtained by determining the iodine content in the antibody using flow injection ICP-MS and iodine inorganic standards for calibration, while the antibody concentration was obtained by UV–VIS DCFH-DA (using an immunoglobulin G standard as cali-brator). The obtained results revealed a stoichiometry of 27.3 ± 0.4 (mean ± standard deviation, SD, n = 3) moles of iodine per mole of antibody. By consulting the antibody sequence in the UniProtKB data-base, it was possible to obtain that the antibody contains 26 tyrosine residues, the amino acid that is more likely iodinated under the pro-posed conditions, as previously reported by other authors [29].
    3.2. Evaluation of the antibody efficiency after labelling
    One of the risks associated to antibody labelling with iodine refers to the loss of antibody recognition capabilities upon reaction. Oxidation damage, specifically the oxidation of methionine to methionine sulf-oxide and tryptophan to the oxindole occurs due to the use of excesses of oxidizing agents that come into intimate contact with the protein in solution. This damage affects the physicochemical integrity of the protein and results in losses of their biological and immunological ac-tivity, especially when the tryptophan or methionine residues are in-volved in the biological or immunological sites of these molecules [30]. Therefore, before using the iodinated antibody for the corresponding application, their reactivity was tested using a transferrin standard as 
    Fig. 3. SEC-UV/VIS (280 nm) chromatograms obtained for transferrin before (blue trace) and after (red trace) immune subtraction using iodinated antibody coupled to AffiAmino Ultrarapid Agarose Magnetic beads. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).
    antigen and conducting quantitative immune affinity subtraction ex-periments. For this aim, we have used the AffiAmino Ultrarapid Agarose magnetic beads (Lab on a bead). They consists of super-para-magnetic agarose beads functionalized with amino-reactive groups and can be used for covalent coupling of molecules with primary amino-groups such as antibodies. The agarose matrix enables minimal non-specific binding of the amino-containing molecules due to its hydro-philic nature. Therefore, the iodinated antibody was first coupled to the beads using the protocol previously described. After blocking the re-maining binding sites on the beads surface, the antibody coupled beads were adequately washed to be used. In order to address the binding efficiency of the iodinated antibody to the beads, aliquots of the anti-body solution (50 µg mL−1) were analyzed by SEC-UV/VIS before and after coupling. Fig. 3 shows both chromatographic profiles obtained at 280 nm (blue trace, before immobilization on the beads and red trace, after immobilization). The evaluation of the data revealed a coupling efficiency of about 72% of the antibody on the beads. r> The next step was the quantitative immune subtraction experiment of transferrin using the magnetic agarose beads coated with the iodi-nated-antitransferrin antibody, as summarized in Fig. 4A. In this case, different concentrations of a transferrin standard (10 and 20 µg mL−1) were prepared in binding buffer (PBS). Aliquots of these solutions were incubated with the iodinated-antitransferrin antibody coupled beads following the previously described procedure. As before, the transferrin standards were analyzed before and after incubation by SEC-UV/VIS to address the remaining species in solution. The experiment was repeated three times for each transferrin concentration. The results obtained revealed a subtraction capability of the iodinated antibody of 94 ± 3% (mean ± SD) for the transferrin standard of 10 µg mL−1 and 96 ± 2% (mean ± SD) for the transferrin standard of 20 µg mL−1. Therefore, after the iodination reaction the antibody does not seem to undergo any detectable losses of their recognition capabilities and can be used for further experiments.
    3.3. Sandwich immunoassay coupled to ICP-MS detection
    In order to conduct the quantitative analysis of transferrin in bio-logical samples with the iodinated antibody in the most sensitive and simple way, a sandwich immunoassay was designed by means of using two different anti-transferrin antibodies: the iodinated one and a sec-ondary biotinylated antibody as summarized in Fig. 4B. The immune complex formed in solution was then captured using streptavidin-coated magnetic microparticles. After washing, to remove unbound substances, the microparticles with the immune-complex were