Irving L. Spar

[22] Iodine monochloride (ICI) iodination techniques

Publisher Summary This chapter describes iodine monochloride (ICI) iodination techniques. The chapter explains that, for labeling proteins and related substances in solution with 131 I or 125 I, the use of the ICI isotopic exchange procedure may often be the method of choice. In this technique, the total amount of iodine incorporated into the iodinated material is known and can be controlled and it avoids the use of oxidizing agents that may damage protein. The basic method, as it is most widely used today, is 131 I or 125 I in the form of iodide ions are mixed with the protein solution to be iodinated, buffered to a pH of approximately eight. As the only oxidizing agent present during the iodination procedure is the ICI itself, no harm can come to protein that would be produced by other oxidizing agents. Also, the ICI method has not been found to be a useful procedure for directly labeling cells or cell membranes; it is useful in preparing reagents for other types of 131 I or 125 I labeling, for example, the preparation of diazotized diodosulfonilic acid for labeling of cell membranes.

131I‐labeled antibodies to human fibrinogen. Diagnostic studies and therapeutic trials

Homologous 131I fibrinogen and its purified antibody will concentrate in a major portion of the neoplasms of the dogs and humans into which they are injected, according to new data. This report gives the scintillation scanning results obtained after iv administration of 131I‐labeled and purified antihuman fibrinogen to 172 consecutive patients with various types of neoplasms. In 75% (129/172) of the cases the tumor, in various sites, was unequivocally located. These included mammary carcinomas (12/17), malignant melanoma (16/26), bronchogenic carcinoma (27/34), osteogenic sarcomas (9/9), hypernephromas (6/6), and primary tumors of the brain (10/18). The 131I deposition in tumor relative to normal tissue and blood was so great that in 12 terminal patients therapeutic trials were initiated using 100 to 160 me of the labeled antibody preparation. In some of these attempts, there was substantial remission of clinical symptoms. The usefulness of this technique for specific radiation therapy of tumors will be greatly improved when methods are developed for increasing 131I concentration in the peoplasm. Several approaches, including fever, endotoxin, pretreatment with fibrinolytic agents and post‐treatment with antifibrinolytic agents, are under intensive investigation in experiments and offer great promise.

Studies Directed Toward the use of Antibodies as Carriers of Radioactivity for Therapy

The relationship of immunological reactions to the growth of normal and abnormal tissues are reviewed. The highly selective localization of a radioactivity-carrying antibody in a particular normal or neoplastic tissue depends on the existence of an antigenic substance accessible to intravenously administered antibody. Procedures likely to be involved in using antibodies for the tissue-specific localization of radioisotopes in therapeutic dosage ranges are discussed.

High Specific Activity Labeling of Protein with I131 by the Iodine Monochloride Method.

Summary A modified iodine monochloride method suitable for preparing I131-labeled proteins of a high degree of radioactivity is described, and results are given. Hydrogen peroxide present in high level I131 preparations is destroyed with catalase. Then IC1 is added to a mixture of the I131 as iodide and the protein to be iodinated. Total iodine content of I131 preparations sets a limit on the specific activity of I131-labeled proteins that can be achieved with a low degree of iodination. In the two commonly used methods for producing I131 (fission of U235 and thermal neutron irradiation of natural tellurium) stable I127 and long-lived I129 are also formed. Analysis showed the total iodine content of fission product I131, as received from Oak Ridge National Laboratory soon after processing, to average 2.4 μg per 100 mc, 3 times the amount present as I131 (0.8 μg/100 mc). For I131 produced from tellurium it was substantially greater. Since the ratio of total iodine to I131 increases with time after processing, freshly produced I131 is necessary to make very high level labeled preparations. Precautions to prevent protein damage as a result of high level labeling procedures are described.

In vivo and in vitro studies of labeled antibodies against rat kidney and Walker carcinoma.

Summary An in vitro method is described for demonstrating the binding by rat organ homogenates of I131 labeled antibodies produced by rabbits immunized with particular rat organs. By this test anti-kidney antibodies show specificity for kidney compared with other rat tissues, and anti-Walker rat carcinoma 256 antibodies specificity for this tumor tissue. These data are compared with I131 distributions found after injection of these antibodies by various routes into living rats. The results suggest that nearly all of these antibody molecules are also general anti-rat antibodies in that they are bound by components of other rat tissues. Such factors as relative circulation rates, blood vessel permeability to proteins, and intracellular or extracellular location of antigens, as well as relative organ specificity of antibodies as demonstrated by in vitro experiments, are probably therefore important in determining the localization of such antibodies in animal injection experiments.

Oxidative iodine monochloride iodination technique

The iodine monochloride (IC1) technique is used to radiolabel proteins under mild experimental conditions. Proteins labeled by this technique have been shown to have both in vitro in vivo characteristics often superior to those proteins labeled by the more frequently used chloramine-T method. Although first published more than two decades ago, use of the IC1 technique has largely been superceded by other more recently developed iodination methods. This paper attempts to reintroduce th oxidative IC1 method to the research community. This is accomplished by the presentation of a detailed protocol for a convenient modification of the original technique. A review of the qualities of the iodinated proteins produced by the IC1 and other iodination methods is also discussed.

Detection of Preformed Venous Thrombi in Dogs by Means of I 131 -Labeled Antibodies to Dog Fibrinogen

Dogs with thrombin-induced thrombi received I131 rabbit antibody to dog fibrinogen and a day later were given antiserum to the rabbit gamma globulin. Scintillation scanning techniques successfully detected the site of thrombosis. Excision of the thrombi and surrounding blood vessels demonstrated deposition of radioactivity in the lesion. The immunologic removal of the gamma globulin from the circulating blood increased the difference between the radioactivity deposited in the lesion and that present in the blood. This accentuates the lesion and offers potential diagnostic advantage.

Localization of I131 Labeled Antibody to Rat Fibrin in Transplantable Rat Lymphosarcoma.

Summary Rabbits were immunized with rat fibrin and the resulting antisera clotted with normal rat plasma. From the clot a substance was isolated and labeled with I131 that showed a strong tendency to bind to rat fibrin clots formed in its presence. This substance, presumably an antibody to rat fibrin, when injected intravenously into rats bearing the transplantable Murphy-Sturm lymphosarcoma localized with a high degree of preferentiality in this tumor.

Localization of intracranial tumors. Comparative study with131I-labeled antibody to human fibrinogen and neohydrin- 203Hg

Two of the most promising isotopically labeled compounds for the localization of intracranial tumors by photoscanning are compared. 203Hg‐neohydrin and 131I‐labeled antibody to human fibrinogen are equal in effectiveness in localizing known tumors in the cranium, and they introduce approximately the same proportion of false information. 203Hg‐neohydrin is more convenient: it is commercially available. Furthermore, scans can be performed as early as four hours after its administration, whereas an interval of at least 40 hours is necessary following the administration of 131I‐labeled antibody before scanning can be efficiently undertaken, because of the slow clearance of this agent from the blood. A 44 to 48 hour interval was used for 131I‐labeled antibody in this study. 131I‐antibody is effective for detecting tumors outside the cranium. For tumors inside the cranium this localization technique with either agent has been found to be extremely useful in neurosurgical care.

Detection of left atrial thrombi

Abstract Scintillation scanning to detect atrial thrombi after intravenous administration of 131 I labeled antibodies to human fibrinogen was performed on 30 patients who had mitral valve surgery under cardiopulmonary bypass. Twenty-six patients had negative scans and at operation no thrombus was found in 25. Of the 4 patients with positive scans, 3 had left atrial thrombi at surgery and 1 had a highly calcified valve. 131 I determinations of blood, surgically removed cardiac tissue, and thrombi demonstrated that the thrombi could have as much as 25 times more 131 I than that in an equivalent amount of blood. Little 131 I accumulation was found in organized, avascular portions of the thrombus or atrial appendage, mitral valve, and tricuspid valve. Because of the high concentration of 131 I in the thrombus compared to that in the cardiac tissue and blood, this procedure seems promising as a means of detecting atrial thrombi with little discomfort to the patient. It is suggested that the use of an antiserum to rabbit gamma globulin to remove immunologically the major portion of the blood-borne 131 I rabbit antiserum to human fibrinogen may appreciably increase the ratio of thrombus to blood and this would decrease the possibility of falsenegative scans and increase the resolution of positive scans.