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125I is a commonly used radioisotope for radio-labeling. This is because 125I is readily available in the form of Na125I with a relatively inexpensive way and has a high specific activity. Na125I emits rays which can readily be detected and quantified with the help of the counter.
However, some precautions must be considered seriously to minimize exposure of workers to the radiation and also to prevent contamination and ingestion. Labeling of antibody involves oxidative generation of cationic I (I+), which is spontaneously added to tyrosine residues and to a lesser extent to the tryptophan and histidine via an electrophilic substitution reaction.
The concentration of the reagents should be maintained in such a way that allow for only one or two tyrosine residues per antibody to be 125I labeled otherwise loss of immunoreactivity damage may occur.
There are other several established methods for radioiodination, which differ in the choice of oxidizing agent used to generate I+. Since strong oxidizing agents can destroy the immunoreactivity of antibodies, there must be a compromise between the efficient generation of I+ and preservation of antigen-binding capacity.
Chloramine-T (N-chloro-p-toluene sulfonamide) is an aromatic oxidizing agent commonly used for the high specific iodination of antibodies and other proteins. Chloramine-T iodination is very rapid, but it can denature antibodies and lead to lose of antigen-binding capacity. The reaction must be stopped quickly after appropriate iodination by adding a reducing agent such as sodium metabisulfite or excess of tyrosine to “mop up” iodine as iodotyrosine.
Following radioiodination, the antibody needs to be separated from free 125I. This can be achieved either by gel filtration or ion exchange chromatography. If IgG antibody has been radiolabeled, ion exchange chromatography can be employed to separate the radiolabeled antibody from free 125I by using a strongly basic anion exchanger that can absorb the iodotyrosine.
The use of chloramines-T immobilized on the beads offers a milder alternative for radioiodination because the reaction can be stopped simply by removing the solid material. Similarly, another agent commonly used for radioiodination is iodogen (1,3,4,6-tetrachloro-3α,6α-diphenylglycoluril), which is even milder than chloramines-T.
Similarly, radioiodination of antibodies using the enzyme lactoperoxidase is a very mild procedure that does minimal damage to antibodies but does not produce radio-labeled antibodies of high specific activity. Lactoperoxidase-catalyzed iodination is therefore used only for iodinating cell surface components since it minimizes the diffusion of reactants across the cell membrane to label internal components.
Lastly, antibodies can also be conjugated with low molecular weight, such as radioiodinated phenolic compounds. Commonly, N-succinimidyl 3-(4-hydroxyphenyl) propionate (the Bolton-Hunter reagent) is radioiodinated using chloramines-T and the 5-[125I] iodophenyl derivative is then conjugated to amino groups of the antibody.