ORIGINAL Annals of Nuclear Medicine Vol. 5, No. 3, 83-87, 1991 Preparation of radioiodinated secretin for radioimmunoassay Chung-Huei HSU,* Kuo-Wei YIN,* Tsai-Hsia HONG,** Tswen-Kei TANG,** Long-Shyong LEE,* Tong H. CHANG,** Jean-Dean LIU*** and Pao-Huei CHEN*** *Division of Nuclear Medicine, Department of Clinical Pathology, Taipei Municipal Jen-Ai Hospital, Taipei, Taiwan, R.O.C. **Cell Biology and Immunology Division, Development Center for Biotechnology, Taipei, Taiwan, R.O.C. ***Department of Internal Medicine, Taipei Municipal Jen-Ai Hospital, Taipei, Taiwan, R.O.C. Radioiodination of synthetic human secretin on its N-terminal histidyl residue was not difficult when a greater amount of Chloramine T and a longer reaction time were employed to achieve better incorporation of 125I. The radioiodinated tracer for an optimal radioimmunoassay required purification. The combination of Sep-pak C18 Cartridge and high performance liquid chromatography for the purification of 125I-secretin in our study revealed that the Sep-pak cartridge was a preliminary step in removing unlabeled radioactive iodide, the reactant, and labeled materials unadsorbed to the cartridge. The eluate eluted from the Sep-pak containing high radioactivity and high immunoreactivity to the antibody were selected for further purification by HPLC which eliminated undesirable radiolabeled substances with lower immunoreactivity. The purified radiolabeled secretin was used in developing a sensitive radioimmunoassay. Key words: radioiodination, secretin, HPLC, radioimmunoassay INTRODUCTION SECRETIN, a 27 amino acid peptide, which contains an N-terminal histidyl and no tyrosyl residue, has been considered to be difficult to use in preparing a satisfactory radioiodine labeled tracer.1,2 A comparative study on 125I-secretin and 125I-6-tyrosyl secretin for radioimmunoassay has been reported.3 For a sensitive radioimmunoassay, the labeled antigen (tracer) and its specific antibody should be small in concentration and great in affinity binding of the immunochemical reaction. The tracer must be purified if the antiserum was obtained from animals immunized with semipurified antigen or was contaminated with non-specific antibody.4-7 The purification procedures are required to remove the unlabeled antigen and labeled antigen with lower Received June 19, 1990, revision accepted October 24, 1990. For reprints contact: Chung-Huei Hsu, M.D., Division of Nuclear Medicine, Department of Clinical Pathology, Taipei Municipal Jen-Ai Hospital, Taipei, Taiwan, R.O.C. immunoreactivity to the antibodies from the reaction mixture.8 MATERIALS AND METHODS Synthetic human secretin was purchased from Peninsula Company, USA. The peptide was diluted to a final concentration of 0.5 mg/ml with 0.01 N HCl and stored at -40deg.C before iodination. Anti-human secretin sera were produced at the Development Center for Biotechnology (DCB), Taipei, Taiwan, by immunizing rabbits repeatedly for 6 months with synthetic human secretin (Peninsula). The antibodies were stored at -70deg.C until use. Na 125I (pH 10 in NaOH) with a specific activity of 17.4 mCi/mg as indicated by the manufacturer was obtained from New England Nuclear Company, Boston, MA, USA. Chloramine T (Trihydrate) (Merck, Darmstadt) and Metabisulfite (Merck, Darmstadt) were prepared at a concentration of 5 mg/ml in 0.25 M sodium phosphate, pH 7.5, and stored in the dark at -40deg.C until use. Sep-pak C18 cartridge and Nova-pak C18 cartridge liquid chromatography columns for use in conjunction with a Waters HPLC purchased from Waters Associates, Milford, MA, USA, were used for the purification of iodinated antigens. The iodination procedures were carried out by a modified Chloramine T method.8 Reagents were added in the following order: 20 ul of 0.25 M sodium phosphate, pH 7.5; 3 ul (300 uCi) of Na 125I; 2 ul of synthetic human secretin; 5 ul of Chloramine T; 10 ul of Metabisulfite was added one minute after the addition of chloramine T. The reaction was accomplished at room temperature. At the end of iodination, 1 ml of blood bank plasma diluted 1: 10 was added to the reaction mixture. The reaction mixture was loaded onto a Sep-pak C18 cartridge, prewashed with 5 ml of 100% ethanol and 5 ml of 0.01 N HCl. After loading the sample, the Sep-pak was washed with 2 ml of distilled water, followed by serial elution of the iodinated substances with 30%, 35%, 40%, 45%, and 50% ethanol in 0.01 N HCl. The elution was performed subsequently with 4 ml of ethanol at each concentration described above and the eluate was collected in a volume of 1 ml per tube. The collected fractions were assessed for their radioactivity (cpm) by counting in a gamma counter (IsoData, Illinois, USA). Identification of immunoreactivity of each fraction was performed by incubating the eluate with 2,500 cpm and the antibody with 1 : 2,000 dilution in 1 ml buffer at 4deg.C overnight. The eluates containing high immunoreactivity and high radioactivity were collected for further HPLC purification. The collected eluate was diluted with 2 volumes of 0.1% Trifluoroacetic acid (TFA) and injected into the Nova-pak C18 cartridge (3.9 mm x 15 mm) column (loop volume 10 ml) and eluted with a gradient of 30%-60% Acetonitrile (ACN) from 20 min to 70 min after injection. The chromatography was run for 80 min at a flow rate of 0.5 ml/min, and the eluate was collected in 1 ml fractions. The eluate of each fraction was assessed for radioactivity and immunoreactivity as described above. The optimal dilution of the antibody was determind by incubating serial dilution of the antibody with the labeled secretin purified by HPLC for 1-3 days at 4deg.C. The inhibition curve of human secretin was assessed by incubating the mixture of the following three substances: (1) 2,000 cpm HPLC purified labeled secretin, (2) antibody with optimal dilution. (3) serial concentrations of synthetic human secretin, in 0.05 M acetate buffer (pH 5.0) containing 0.2% BSA as buffer. The final volume of the incubation mixture was 1 ml per tube. The adsorption of free peptides to 1% Dextran T 70 coated charcoal was used as the separation method. The equilibrium and non-equilibrium systems were investigated. RESULTS The efficiency of iodination was approximately ranged from 20%-30%, demonstrated by the percentage of adsorbed radioactivity on the Sep-pak C18 cartridge. The effect of the Sep-pak C18 cartridge used in purifying radiolabeled antigen was rapid and the results were reproducible. It removed the unlabeled radioactive iodide, nonradioactive materials, and labeled substances which had no immunoreactivity with the antibodies, by selectively adsorbing and eluting with different concentrations of ethanol. It provided an effective and efficient means of preparing the labeled peptide for further purification. There were four main radioactivity peaks, eluted at 35%, 40%, 45% and 50% ethanol/0.01 N HCl, containing about 34%, 29%, 26%, and 9% of the total radioactivity adsorbed to the Sep-pak cartridge, respectively (Fig. 1). The immunoreactivity of each fraction is also shown in the figure. Further HPLC purification of the eluate eluted from 40% ethanol (fraction 40-2, 40-3, 40-4 were pooled) and that from 45% ethanol (fraction 45-1, 45-2, 45-3 were pooled) both revealed two radioactive peaks of eluate which were eluted by 44% and 50% of ACN, respectively (Fig. 2). The immunoreactivity existed only in the eluate of the first peak and the immunoreactivity was much higher in the eluate which was pretreated with Sep-pak eluted from 45% ethanol than that from 40% ethanol, as also shown in the figure. The dilution of the antibody was optimal in 1 : 50,000 when the antibody was equilibriumly incubated with the eluate in fraction 23 purified with HPLC for 2 days at 4deg.C. The material used for the above HPLC purification was the eluate eluted from 45% ethanol with Sep-pak. The standard curve was constructed by employing the HPLC purified 125I-secretin as tracer in non-equilibrium system with 3 days of preincubation of the antibody with the cold antigen, followed by 2 days of incubation with the labeled antigen. The detecting limit of the assay was 10 pg/ml/tube (Fig. 3). The shelf-life of the labeled preparation was approximately 30 days of storage at -20deg.C. DISCUSSION Because of the lack of tyrosin residue on the secretin, 6-Tyr-secretin was used as an antigen in preparing the radiolabeling tracer. However, the disadvantage in the use of 125I-6-Tyr-secretin for a radioimmunoassay is its diminished immunoreactivity compared to 125I-secretin. Comformational change in the secretin molecule might render it less immunoreactive with antibodies developed in response to secretin immunization.3 The maximal transfer of 125I onto 6-Tyr-secretin occurred within two seconds, whereas a longer time was needed for synthetic human secretin. The efficiency of iodination on either antigen was not significantly different.3 Radioimmunoassay is an immunochemical procedure, based on the competing reaction between the unlabeled and labeled antigens (tracer) for binding to specific antibodies. The labeled antigen must be highly purified to avoid the interaction of labeled contaminants with the nonspecific antibodies. For a sensitive radioimmunoassay we need to use a low concentration of the tracer, preparing a labeled hormone with high specific activity and high affinity for specific antibodies. The procedures are required to remove the unreacted radioiodide, the reactants, and the damaged iodinated protein with lower immunoreactivity from the reaction mixture. Several conventional separation methods such as gel filtration, dialysis, ion-exchange chromatography, adsorption chromatography, and gel electrophoresis were used.2,3,10-12 They were not complete and were time consuming. The profiles from our combination of Sep-pak and HPLC studies suggest that the Sep-pak cartridge was a preliminary step in removing unlabeled radioactive iodide and labeled materials not adsorbed to the cartridge. The adsorbed materials were separated into four main radioactivity peaks. Immunoreaction with the antibody existed mainly in the eluate eluted from 40% and 45% ethanol. Further purification of the mixture of 45-1, 45-2, and 45-3 with HPLC eliminated about 45% of the radiolabeled substance which did not immunoreact with our antibody. The second peak of eluate might contain damaged iodinated secretin bound to plasma protein which resulted in a loss of immunoreactivity. Antibody dilution test evaluated with the two iodinated mixtures, Sep-pak treated 45-3 and HPLC treated fraction 23 revealed that the ideal B/F for radioimmunoassay, between 0.75 to 1.5, could be obtained when 1 : 50,000 diluted antibody reacted with fraction 23 purified by HPLC incubated for 2 days at 4deg.C, while only 0.5 (B/F) could be reached when 1 : 2,000 diluted antibody reacted with the Sep-pak purified fraction 45-3 under the same incubation conditions. The standard curve constructed with the labeled secretin purified by HPLC would be more sensitive than with that obtained from Sep-pak purified alone. The present report revealed that high performance liquid chromatography was a rapid and applicable method for purification of radiolabeled synthetic human secretin which would be useful in developing a more sensitive secretin radioimmunoassay. ACKNOWLEDGMENTS We like to thank Mr. Sheng-Chang Wang and Shui-Tsung Chen of Development Center for Biotechnology, Taipei, Taiwan, for their technical assistance in antibody production. We also thank Mr. Jenn-Su Lee and Miss Tsuey-Huey Shiau for Manuscript preparation. REFERENCES 1. Straus E: Special problems of the Radioimmunoassay for Gut Hormones. Clin Gastroenterol 9 (3): 555-565, 1980 2. Tai HH, Korsch B, Chey WY: Preparation of I-125 Labeled Secretin of High Specific Radioactivity. Anal Biochem 69: 34-42, 1975 3. Straus E, Urbach HJ, Yalow RS: Comparative reactivities of I-125-secretin and I-125-6-tyrosyl secretin with guinea pig and rabbit anti-secretin sera. Biochem Biophys Res Commun 64 (3): 1036-1040, 1975 4. Yalow RS, Berson SA: General aspects of radioimmunoassay procedures. Proceedings of Symposium of In Vitro Procedures with Radioisotopes in Medicine. IAEA-SM-124/106, Vienna: 455-479, 1970 5. Yalow RS: Radioimmunoassay. Ann Rev Biophys Bioeng 9: 327-345, 1980 6. Yalow RS: Radioimmunoassay. practices and pitfalls. Proceedings AM. Heart Association Council for High Blood Pressure Research. Circ Res 32: I116-I128. 1973 7. Yalow RS: Radioimmunoassay in laboratory medicine. Jpn J Clin Path 32: 490-505, 1984 8. Chervu LR, Murty DRK: Radiolabeling of antigens: Procedure and assessment of properties. Semin Nucl Med 5: 157-172, 1975 9. Hunter WN, Greenwood FC: Preparation of Iodine-131 labelled growth hormone of high specific activity. Nature 194: 495-496, 1962 10. Greenwood FC, Hunter WN, Glover JS: The preparation of I-131 labelled human growth hormone of high specific radioactivity. Biochem J 89: 114-123, 1963 11. Schaffalitzky OB, Muckadell DE, Fahrenkrug J: Radioimmunoassay of Secretin in Plasma. Scand J Clin Lab Invest 37: 155-162, 1977 12. Boden G, Chey WY: Preparation and specificity of antiserum to synthetic secretin and its use in a radioimmunoassay. Endocrinology 92: 1617-1624, 1973