Radioiodination of Antibodies
Antibodies are usually radiolabelled in order to characterise the interaction of an antibody with its specific epitope. This characterisation may consist of the localisation of the interaction, e.g., on a Western blot or an immunoscintigraphic image, or alternatively the quantification of the interaction, e.g., in radioimmunoassay. The radionuclide employed and the technique used to incorporate it into the antibody depend on the application envisaged and the means of localisation and/or quantification. Both the in vitro and the in vivo uses of labelled antibodies span more than 50 years and, during this time, methods for labelling antibodies with at least 20 diffferent radionuclides have been developed. A detailed description of all the techniques used to incorporate all of these radioisotopes is not possible within the space limitations of this book and therefore this article is restricted to the techniques likely to be of interest to the majority of its readership, i.e., radioiodination of antibodies for in vitro applications. A more comprehensive review of methods for labelling antibodies with a wider range of radionuclides can be found in Mather (2000).
II. MATERIALS AND INSTRUMENTATION
The antibody to be labelled must be available in a purified form (any contaminating proteins will also be radiolabelled). The antibody concentration should be 100µg-5mg/ml in either 0.1M phosphate buffer, pH 7.4, or borate, phosphate, or HEPES buffer, pH 8, as indicated in the procedures.
Radioisotopes are available from Amersham Biosciences: Radioiodine Na125I (100mCi/ml 3.7GBq/ml) (IMS30 Amersham), N-Succimidyl-3-(4-hydroxy-3- [125I]iodophenyl)propionate, Bolton and Hunter reagent (IM5861). Unless otherwise indicated, all reagents are from Sigma Aldrich Chemical Company, Poole, UK: chloramine-T (40,286-9), sodium metabisulphite ($9000), disodium hydrogen phosphate (21,988- 6), sodium dihydrogen phosphate (S8282), Iodogen (Pierce Chemical Company, 28,600), dichloromethane (43,922-3), methanol (44,347-6), Sephadex-G50 fine grade (G-50-80), prepacked PD-10 column (Amersham Biosciences 17-0851-01), bovine serum albumin (BSA) (A7906), silica gel-coated plastic TLC sheets (Polygram 805013, Marchery-Hagel, Duren, Germany), silica gelimpregnated glass fibre (ITLC) sheets (61,885, Pall Corp., Ann Arbor, MI), and Whatman 3 MM Chr paper (Z27,085-7).
Many alternatives exist for the equipment used for these procedures. The following are the ones used in this laboratory: Gamma counter (LKB Ultragamma, Wallac, Finland), vortex mixer (MS2 minishaker, Staufen, Germany), microcentrifuge (MSE Microcentaur, Crawley, UK), rotamixer (Denley Spiramix, Thermo Life Sciences, Basingstoke, England), and Savant Speed-Vac (Westwood, MA).
All radiolabelling procedures must be performed with attention to good radiation safety practice. In particular, radioiodinations should be performed in a well-ventilated fume hood and all containers should be locally shielded by keeping them in small lead pots. A lead-glass L-shield should be used to reduce wholebody radiation doses.
A. Radioiodination with Iodine- 125 Using Chloramine-T as Oxidant
This procedure is adapted from that originally published by Hunter and Greenwood (1962).
B. Radioiodination with Iodine-125 Using lodogen as Oxidant
This procedure is adapted from that originally published by Fraker and Speck (1978).
C. Iodination of Antibody with "Bolton and Hunter" Reagent
This procedure is adapted from that originally published by Bolton and Hunter (1973).
D. Separation of Radiolabelled Antibody from Free Iodide
E. Determination of Radiochemical Purity by TLC
85% methanol solution: Pour 85 ml of methanol into a measuring cylinder and make up to 100ml with deionised water. Store in a tightly closed container at room temperature for up to 4 weeks.
F. Measurement of the Immunoreactive Fraction of Radiolabelled Antibody
This assay has been adapted from a method published by Lindmo et al. (1984). Modifications include a number of simplifications that make the assay easier and quicker but which could correctly be criticised if used out of context. This modified assay is, therefore, only recommended as a "quality control check" rather than as a way of determining the real immunoreactive fraction of the antibody for which the original published method is recommended.
Many techniques have been developed in the last 50 years for labelling proteins with radioiodine but, for various reasons, most of these are now only of academic interest, at least so far as the routine labelling of antibodies is concerned. For the interested reader, a detailed review has been put together by Dewanjee (1992). The methods practised most widely are those in which the radioiodine is oxidised to a reactive intermediate positively charged species such as the hydrated iodinium ion H2OI+, which then reacts via electrophilic substitution for the activated protons on the phenolic ring of the tyrosine side chains. These iodinium species or related cations can be produced by reacting the radioiodide with a variety of oxidising agents, but two in particular have become the most popular: chloramine-T (N-chloro-p-toluenesulphonamide) and Iodogen (diphenylglycoluril). Methods for labelling antibodies with radioiodine using these two methods were described in Sections III,A and III,B. Both techniques have their own inherent advantages and drawbacks. The Iodogen method (Fraker and Speck, 1978) is extremely simple and largely invariable. The concentration of the oxidant is determined by its very poor solubility in aqueous solvents and therefore the only variables one can change in order to influence the reaction are incubation time and temperature. Even so, this method normally produces very acceptable labelling efficiencies of the order of 80-95%. In contrast, details of the chloramine-T method (Hunter and Greenwood, 1962) vary widely from laboratory to laboratory. Different researchers have their own favoured oxidant concentrations, incubation times, and choice of quenching reagents. This method has the (somewhat theoretical) advantage that it can be tailored for different proteins, but has the disadvantage that the reagents have to be freshly prepared prior to use and overenthusiastic attempts to improve labelling efficiencies with high concentrations of the oxidant can lead to antibody damage.
Only in rare circumstances will either labelling procedure result in labelling efficiencies approaching 100%. The consequence of this is that the reaction mixture will contain a significant proportion of unreacted "free iodide." It is normally desirable to remove this free iodine in order to provide a preparation with sufficiently high radiochemical purity. The most widely used method for the purification of labelled antibody preparations is size-exclusion chromatography on a short Sephadex column as described in Section III,D, but several alternatives, such as ionexchange chromatography or the use of spin columns, exist, some of which are potentially more convenient. Before and after purification of the labelled antibody it is useful to measure the purity of the labelled antibody preparation, initially to check the efficiency of the labelling procedure and then later to determine the purity of your reagent. A very simple means of measuring this purity, based on ascending thin-layer chromatography, is described in Section III,E.
The main reason for labelling an antibody is to obtain a radioactive molecule that binds to a specific recognition site. It is therefore essential that the antibody retains the ability to bind to its epitope throughout the labelling procedure. The best tests to ensure that this is the case are radioligand binding assays, which measure directly the binding of the radiolabelled molecules rather than the whole population of antibody molecules in solution, most of which will not be labelled. These assays fall into two categories: those used to determine the binding affinity of the antibody and those intended to measure the proportion of labelled molecules that retain some ability to bind specifically to their epitope. For the purposes of a relatively simple check to see if the antibody remains functional after labelling, the latter type of assay is the more appropriate and a protocol describing such a test can be found in Section III,F.
Two main types of mechanism can compromise the immunoreactivity of a labelled antibody. The first is due to the effect of the steric hindrance of the large iodine atom when it is substituted into a critical tyrosine residue close to the binding site of the antibody. Although the radioiodine can potentially react with any of several tyrosine amino acids scattered throughout the antibody molecule, factors such as local charge distribution and accessibility mean that one or more residues will be labelled preferentially. If one of these sites happens to be in one of the critical CDRs, then a significant degree of antibody binding will be lost. If this happens, the number of possibilities for solving the problem are limited. It is possible that a change in pH during the labelling procedure may alter the local charge distribution and favour an alternative tyrosine residue, albeit at the risk of a lower labelling efficiency. If this does not work, then the only alternative is to use an entirely different chemistry for radioiodination. The most well-established alternative is the Bolton and Hunter method described in Section III,C, which results in antibody labelling at the site of lysine residues.
The other type of mechanism responsible for loss of immunoreactivity is oxidation. In addition to its desired role in oxidising the radioiodide to a reactive species, the oxidant may potentially oxidise critical residues, particularly methionine, in the antibody molecule. A way to find out which of the two possible mechanisms may in fact be the cause of a loss in immunoreactivity is to perform the labelling procedure without the addition of the radioiodine and to perform an ELISA assay. If an oxidative mechanism is responsible, then immunoreactivity will still be lost, as all the antibody molecules will be affected, not only those substituted with radioiodine. If this is found to be the case then either a Bolton and Hunter approach can be pursued or an alternative electrophilic substitution method that does not subject the antibody to such strong oxidising conditions can be employed. Two approaches may work. The first is to use a milder oxidising agent, such as the lactoperoxidase system (Morrison and Bayse, 1970). The alternative is to use a modification of the Iodogen system in which the radioiodine is first oxidised in the iodogen tube but is then transferred from the oxidising environment to another tube containing the antibody (van der Laken et al., 1997). It is likely that both of these procedures will result in a lower labelling efficiency but either may solve the problem of oxidative damage to the antibody.
The shelf life of radioiodinated antibodies is limited by radiolysis, which causes a gradual loss in both purity and immunoreactivity. The rate of deterioration can be reduced by the addition of carrier proteins or antioxidants that scavenge the radiolytic free radicals (Chakrabarti et al., 1996). A concentration of 0.1-1% albumin or 0.5% ascorbic acid is commonly used and antibodies may be stored in these solutions at either 4 or -20°C for at least a month without a significant loss of quality. If stored below 0° then the preparation should be divided into aliquots to save repeated freezing and thawing, which tends to favour aggregation of the antibody. If stored above 0° provided it does not interfere with the ultimate application, sodium azide can be added to a final concentration of 0.05% to limit microbial growth.
The most likely cause of failure of any of the labelling methods described here is the presence of impurities in the antibody solution. The best solution is to repurify the antibody by either dialysis or gel filtration into freshly prepared buffers.
The most common problem experienced with the immunoreactive fraction assay described in Section III,F is that a curve, rather than a straight line, is obtained when data are plotted. This is nearly always caused by inaccuracies in diluting and losses in washing the cells. With practice and care, the problem usually goes away.
Bolton, A. E., and Hunter, W. M. (1973). The labelling of proteins to high specific activities by conjugation to a 125-I-containing acylating agent. Biochem. J. 133, 529-538.
Chakrabarti, M. C., Le, N., Paik, C. H., De Graft, W. G., and Carrasquillo, J. A. (1996). Prevention of radiolysis of monoclonal antibody during labeling. J. Nuclear Med. 37(8), 1384-1388.
Dewanjee, M. K. (1992). Radioiodination: Theory, Practice and Biomedical Applications. Kluwer Academic, Dordrecht.
Fraker, P. J., and Speck, J. C. (1978). Protein and cell membrane iodinations with a sparingly soluble chloramide 1,3,4,6-tetrachloro 3a.6a diphenylglycoluril. Biochem. Biophys. Res. Commun. 80, 849.
Hunter, W. M., and Greenwood, E C. (1962). Preparation of iodine- 131 labelled human growth hormone of high specific activity. Nature 194, 495-496.
Lindmo, T., Boven, E., and Cuttita, E (1984). Determination of the immunoreactive fraction of radiolabelled monoclonal antibody by linear extrapolation to binding at infinite antigen excess. J. Immunol Methods 27, 77-89.
Mather, S. (2000). Radiolabelling of monoclonal antibodies. In "Monoclonal Antibodies, a Practical Approach" (P. Shepherd and C. Dean, eds.), pp. 207-236. Oxford Univ. Press, Oxford.
Morrison, M., and Bayse, G. S. (1970). Catalysis of iodination by lactoperoxidase. Biochemistry 9, 2995-3000.
van der Laken, C. J., Boerman, O. C., Oyen, W. J., van de Ven, M. T., Chizzonite, R., Corstens, F. H., and van der Meer, J. (1997). Preferential localization of systemically administered radiolabeled interleukinl alpha in experimental inflammation in mice by binding to the type II receptor. J. Clin. Invest. 100(12), 2970-2976.
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